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  • Tfap2a and 2b act downstream of Ptf1a to promote amacrine cell differentiation during retinogenesis. 25966682

    Retinogenesis is a precisely controlled developmental process during which different types of neurons and glial cells are generated under the influence of intrinsic and extrinsic factors. Three transcription factors, Foxn4, RORβ1 and their downstream effector Ptf1a, have been shown to be indispensable intrinsic regulators for the differentiation of amacrine and horizontal cells. At present, however, it is unclear how Ptf1a specifies these two cell fates from competent retinal precursors. Here, through combined bioinformatic, molecular and genetic approaches in mouse retinas, we identify the Tfap2a and Tfap2b transcription factors as two major downstream effectors of Ptf1a. RNA-seq and immunolabeling analyses show that the expression of Tfap2a and 2b transcripts and proteins is dramatically downregulated in the Ptf1a null mutant retina. Their overexpression is capable of promoting the differentiation of glycinergic and GABAergic amacrine cells at the expense of photoreceptors much as misexpressed Ptf1a is, whereas their simultaneous knockdown has the opposite effect. Given the demonstrated requirement for Tfap2a and 2b in horizontal cell differentiation, our study thus defines a Foxn4/RORβ1-Ptf1a-Tfap2a/2b transcriptional regulatory cascade that underlies the competence, specification and differentiation of amacrine and horizontal cells during retinal development.
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  • Astrocytes in the rat nucleus tractus solitarii are critical for cardiovascular reflex control. 24259582

    We have shown that an antibody to dopamine-β-hydroxylase conjugated with saporin (anti-DBH-SAP) damages catecholamine neurons in the nucleus tractus solitarii (NTS) of rat, attenuates arterial baroreflexes, and leads to lability of arterial blood pressure, damage to cardiac myocytes, and, in some animals, sudden death. However, others have shown that injection of 6-hydroxydopamine (6-OHDA), a toxin devoid of saporin, also damaged NTS catecholamine neurons but did not lead to these cardiovascular changes. We found similar cardiovascular changes after injecting a different SAP conjugate to target NTS neurons with neurokinin (NK1) receptors. Because ribosome-inactivating proteins may be toxic to glia, we hypothesized that SAP, a ribosome-inactivating protein, might target glia whose loss could account for physiological changes. We tested this hypothesis by assessing effects on select neurons and on glia in the NTS after exposure to SAP, targeted SAP conjugates, or 6-OHDA. SAP and all SAP conjugates led to loss of immunoreactivity for glial fibrillary acidic protein, a marker for astrocytes, in the NTS while 6-OHDA did not. As reported previously, anti-DBH-SAP selectively killed noradrenergic neurons in the NTS while SAP conjugated to stabilized substance P (SSP-SAP) selectively killed neurons with NK1 receptors. In contrast, SAP produced no demonstrable neuronal damage. All injections led to activation of microglia in the NTS; however, only SAP and its conjugates attenuated cardiovascular reflexes while also producing lability of arterial pressure, damage to cardiac myocytes, and in some animals, sudden death. Thus, NTS astrocytes may play a role in mediating cardiovascular reflex transmission through the NTS.
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  • Induction of pluripotent stem cells from human third molar mesenchymal stromal cells. 20595386

    The expression of four transcription factors (OCT3/4, SOX2, KLF4, and MYC) can reprogram mouse as well as human somatic cells to induced pluripotent stem (iPS) cells. We generated iPS cells from mesenchymal stromal cells (MSCs) derived from human third molars (wisdom teeth) by retroviral transduction of OCT3/4, SOX2, and KLF4 without MYC, which is considered as oncogene. Interestingly, some of the clonally expanded MSCs could be used for iPS cell generation with 30-100-fold higher efficiency when compared with that of other clonally expanded MSCs and human dermal fibroblasts. Global gene expression profiles demonstrated some up-regulated genes regarding DNA repair/histone conformational change in the efficient clones, suggesting that the processes of chromatin remodeling have important roles in the cascade of iPS cells generation. The generated iPS cells resembled human embryonic stem (ES) cells in many aspects, including morphology, ES marker expression, global gene expression, epigenetic states, and the ability to differentiate into the three germ layers in vitro and in vivo. Because human third molars are discarded as clinical waste, our data indicate that clonally expanded MSCs derived from human third molars are a valuable cell source for the generation of iPS cells.
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  • Decreased expression of neuronal nitric oxide synthase in the nucleus tractus solitarii inhibits sympathetically mediated baroreflex responses in rat. 22687614

    Despite numerous studies it remains controversial whether nitric oxide (NO·) synthesized by neuronal NOS (nNOS) plays an excitatory or inhibitory role in transmission of baroreflex signals in the nucleus tractus solitarii (NTS). In the current studies we sought to test the hypothesis that nNOS is involved in excitation of baroreflex pathways in NTS while excluding pharmacological interventions in assessing the influence of nNOS. We therefore developed, validated and utilized a short hairpin RNA (shRNA) to reduce expression of nNOS in the NTS of rats whose baroreflex activity was then studied. We demonstrate downregulation of nNOS through transduction with adeno-associated virus type 2 (AAV2) carrying shRNA for nNOS. When injected bilaterally into NTS AAV2nNOSshRNA significantly reduced reflex tachycardic responses to acute hypotension while not affecting reflex bradycardic responses to acute increases of arterial pressure. Control animals treated with intravenous propranolol to block sympathetically mediated chronotropic responses manifested the same baroreflex responses as animals that had been treated with AAV2nNOSshRNA. Neither AAV2 eGFP nor AAV2nNOScDNA affected baroreflex responses. Blocking cardiac vagal influences with atropine similarly reduced baroreflex-mediated bradycardic responses to increases in arterial pressure both in control animals and in those treated with AAV2nNOSshRNA. We conclude that NO· synthesized by nNOS in the NTS is integral to excitation of baroreflex pathways involved in reflex tachycardia, a largely sympathetically mediated response, but not reflex bradycardia, a largely parasympathetically mediated response. We suggest that, at the basal state, nNOS is maximally engaged. Thus, its upregulation does not augment the baroreflex.
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  • CDNF protects the nigrostriatal dopamine system and promotes recovery after MPTP treatment in mice. 21943517

    Cerebral dopamine neurotrophic factor (CDNF) is a recently discovered protein, which belongs to the evolutionarily conserved CDNF/MANF family of neurotrophic factors. The degeneration of dopamine neurons following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment is well characterized, and efficacy in this model is considered a standard criterion for development of parkinsonian therapies. MPTP is a neurotoxin, which produces parkinsonian symptoms in humans and in C57/Bl6 mice. To date, there are no reports about the effects of CDNF on dopamine neuron survival or function in the MPTP rodent model, a critical gap. Therefore, we studied whether CDNF has neuroprotective and neurorestorative properties for the nigrostriatal dopamine system after MPTP injections in C57/Bl6 mice. We found that bilateral striatal CDNF injections, given 20 h before MPTP, improved horizontal and vertical motor behavior. CDNF pretreatment increased tyrosine hydroxylase (TH) immunoreactivity in the striatum and in the substantia nigra pars reticulata (SNpr), as well as the number of TH-positive cells in substantia nigra pars compacta (SNpc). Posttreatment with CDNF, given 1 week after MPTP injections, increased horizontal and vertical motor behavior of mice, as well as dopamine fiber densities in the striatum and the number of TH-positive cells in SNpc. CDNF did not alter any of the analyzed dopaminergic biomarkers or locomotor behavior in MPTP-untreated animals. We conclude that striatal CDNF administration is both neuroprotective and neurorestorative for the TH-positive cells in the nigrostriatal dopamine system in the MPTP model, which supports the development of CDNF-based treatment for Parkinson's disease.
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  • Dynamic mass redistribution assay decodes differentiation of a neural progenitor stem cell. 22885730

    Stem cells hold great potential in drug discovery and development. However, challenges remain to quantitatively measure the functions of stem cells and their differentiated products. Here, we applied fluorescent imaging, quantitative real-time PCR, and label-free dynamic mass redistribution (DMR) assays to characterize the differentiation process of the ReNcell VM human neural progenitor stem cell. Immunofluorescence imaging showed that after growth factor withdrawal, the neuroprogenitor stem cell was differentiated into dopaminergic neurons, astrocytes, and oligodendrocytes, thus creating a neuronal cell system. High-performance liquid chromatography analysis showed that the differentiated cell system released dopamine upon depolarization with KCl. In conjunction with quantitative real-time PCR, DMR assays using a G-protein-coupled receptor agonist library revealed that a subset of receptors, including dopamine D(1) and D(4) receptors, underwent marked alterations in both receptor expression and signaling pathway during the differentiation process. These findings suggest that DMR assays can decode the differentiation process of stem cells at the cell system level.
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  • Dopamine neuron glutamate cotransmission: frequency-dependent modulation in the mesoventromedial projection. 19729052

    Mesoventromedial dopamine neurons projecting from the medial ventral tegmental area to the ventromedial shell of the nucleus accumbens play a role in attributing incentive salience to environmental stimuli that predict important events, and appear to be particularly sensitive to the effects of psychostimulant drugs. Despite the observation that these dopamine neurons make up almost the entire complement of neurons in the projection, stimulating their cell bodies evokes a fast glutamatergic response in accumbens neurons. This is apparently due to dopamine neuron glutamate cotransmission, suggested by the extensive coexpression of vesicular glutamate transporter 2 (VGLUT2) in the neurons. To examine the interplay between the dopamine and glutamate signals, we used acute quasi-horizontal brain slices made from DAT-YFP mice in which the intact mesoventromedial projection can be visualized. Under current clamp, when dopamine neurons were stimulated repeatedly, dopamine neuron glutamate transmission showed dopamine-mediated facilitation, solely at higher, burst-firing frequencies. Facilitation was diminished under voltage clamp and flipped to inhibition by intracellular Cs(+) or GDPbetaS, indicating that it was mediated postsynaptically. Postsynaptic facilitation was D1 mediated, required activation of NMDA receptors and closure of voltage gated K(+)-channels. When postsynaptic facilitation was blocked, D2-mediated presynaptic inhibition became apparent. These counterbalanced pre- and postsynaptic actions determine the frequency dependence of dopamine modulation; at lower firing frequencies dopamine modulation is not apparent, while at burst firing frequency postsynaptic facilitation dominates and dopamine becomes facilitatory. Dopamine neuron glutamate cotransmission may play an important role in encoding the incentive salience value of conditioned stimuli that activate goal-directed behaviors, and may be an important subtract for enduring drug-seeking behaviors.
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  • Tbr2 deficiency in mitral and tufted cells disrupts excitatory-inhibitory balance of neural circuitry in the mouse olfactory bulb. 22745484

    The olfactory bulb (OB) is the first relay station in the brain where odor information from the olfactory epithelium is integrated, processed through its intrinsic neural circuitry, and conveyed to higher olfactory centers. Compared with profound mechanistic insights into olfactory axon wiring from the nose to the OB, little is known about the molecular mechanisms underlying the formation of functional neural circuitry among various types of neurons inside the OB. T-box transcription factor Tbr2 is expressed in various types of glutamatergic excitatory neurons in the brain including the OB projection neurons, mitral and tufted cells. Here we generated conditional knockout mice in which the Tbr2 gene is inactivated specifically in mitral and tufted cells from late embryonic stages. Tbr2 deficiency caused cell-autonomous changes in molecular expression including a compensatory increase of another T-box member, Tbr1, and a concomitant shift of vesicular glutamate transporter (VGluT) subtypes from VGluT1 to VGluT2. Tbr2-deficient mitral and tufted cells also exhibited anatomical abnormalities in their dendritic morphology and projection patterns. Additionally, several non-cell-autonomous phenotypes were observed in parvalbumin-, calbindin-, and 5T4-positive GABAergic interneurons. Furthermore, the number of dendrodendritic reciprocal synapses between mitral/tufted cells and GABAergic interneurons was significantly reduced. Upon stimulation with odorants, larger numbers of mitral and tufted cells were activated in Tbr2 conditional knockout mice. These results suggest that Tbr2 is required for not only the proper differentiation of mitral and tufted cells, but also for the establishment of functional neuronal circuitry in the OB and maintenance of excitatory-inhibitory balance crucial for odor information processing.
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  • Generation of a transgenic zebrafish model of Tauopathy using a novel promoter element derived from the zebrafish eno2 gene. 17897967

    The aim of this study was to isolate cis-acting regulatory elements for the generation of transgenic zebrafish models of neurodegeneration. Zebrafish enolase-2 (eno2) showed neuronal expression increasing from 24 to 72 h post-fertilization (hpf) and persisting through adulthood. A 12 kb eno2 genomic fragment, extending from 8 kb upstream of exon 1 to exon 2, encompassing intron 1, was sufficient to drive neuronal reporter gene expression in vivo over a similar time course. Five independent lines of stable Tg(eno2 : GFP) zebrafish expressed GFP widely in neurons, including populations with relevance to neurodegeneration, such as cholinergic neurons, dopaminergic neurons and cerebellar Purkinje cells. We replaced the exon 2-GFP fusion gene with a cDNA encoding the 4-repeat isoform of the human microtubule-associated protein Tau. The first intron of eno2 was spliced with high fidelity and efficiency from the chimeric eno2-Tau transcript. Tau was expressed at approximately 8-fold higher levels in Tg(eno2 : Tau) zebrafish brain than normal human brain, and localized to axons, neuropil and ectopic neuronal somatic accumulations resembling neurofibrillary tangles. The 12 kb eno2 promoter drives high-level transgene expression in differentiated neurons throughout the CNS of stable transgenic zebrafish. This regulatory element will be useful for the construction of transgenic zebrafish models of neurodegeneration.
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  • Testosterone regulation of sex steroid-related mRNAs and dopamine-related mRNAs in adolescent male rat substantia nigra. 22867132

    Increased risk of schizophrenia in adolescent males indicates that a link between the development of dopamine-related psychopathology and testosterone-driven brain changes may exist. However, contradictions as to whether testosterone increases or decreases dopamine neurotransmission are found and most studies address this in adult animals. Testosterone-dependent actions in neurons are direct via activation of androgen receptors (AR) or indirect by conversion to 17β-estradiol and activation of estrogen receptors (ER). How midbrain dopamine neurons respond to sex steroids depends on the presence of sex steroid receptor(s) and the level of steroid conversion enzymes (aromatase and 5α-reductase). We investigated whether gonadectomy and sex steroid replacement could influence dopamine levels by changing tyrosine hydroxylase (TH) protein and mRNA and/or dopamine breakdown enzyme mRNA levels [catechol-O-methyl transferase (COMT) and monoamine oxygenase (MAO) A and B] in the adolescent male rat substantia nigra. We hypothesized that adolescent testosterone would regulate sex steroid signaling through regulation of ER and AR mRNAs and through modulation of aromatase and 5α-reductase mRNA levels.We find ERα and AR in midbrain dopamine neurons in adolescent male rats, indicating that dopamine neurons are poised to respond to circulating sex steroids. We report that androgens (T and DHT) increase TH protein and increase COMT, MAOA and MAOB mRNAs in the adolescent male rat substantia nigra. We report that all three sex steroids increase AR mRNA. Differential action on ER pathways, with ERα mRNA down-regulation and ERβ mRNA up-regulation by testosterone was found. 5α reductase-1 mRNA was increased by AR activation, and aromatase mRNA was decreased by gonadectomy.We conclude that increased testosterone at adolescence can shift the balance of sex steroid signaling to favor androgenic responses through promoting conversion of T to DHT and increasing AR mRNA. Further, testosterone may increase local dopamine synthesis and metabolism, thereby changing dopamine regulation within the substantia nigra. We show that testosterone action through both AR and ERs modulates synthesis of sex steroid receptor by altering AR and ER mRNA levels in normal adolescent male substantia nigra. Increased sex steroids in the brain at adolescence may alter substantia nigra dopamine pathways, increasing vulnerability for the development of psychopathology.
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