Search

The mammalian cochlea undergoes a highly dynamic process of growth and innervation during development. This process includes spiral ganglion neuron (SGN) branch refinement, a process whereby Type I SGNs undergo a phase of “debranching” before forming unramified synaptic contacts with inner hair cells. UsingSox2CreERT2andR26RtdTomatoas a strategy to genetically label individual SGNs in mice of both sexes, we report on both a time course of SGN branch refinement and a role for P2rx3 in this process. P2rx3 is an ionotropic ATP receptor that was recently implicated in outer hair cell spontaneous activity and Type II SGN synapse development (Ceriani et al., 2019), but its function in Type I SGN development is unknown. Here, we demonstrate that P2rx3 is expressed by Type I SGNs and hair cells during developmental periods that coincide with SGN branching refinement.P2rx3null mice show SGNs with more complex branching patterns on their peripheral synaptic terminals and near their cell bodies around the time of birth. Loss ofP2rx3does not appear to confer general changes in axon outgrowth or hair cell formation, and alterations in branching complexity appear to mostly recover by postnatal day (P)6. However, when we examined the distribution of Type I SGN subtypes using antibodies that bind Calb2, Calb1, and Pou4f1, we found thatP2rx3null mice showed an increased proportion of SGNs that express Calb2. These data suggest P2rx3 may be necessary for normal Type I SGN differentiation in addition to serving a role in branch refinement.

SUMMARY Efficient and homogeneous in vitro generation of peripheral sensory neurons may provide a framework for novel drug screening platforms and disease models of touch and pain. We discover that, by ovesssrexpressing NGN2 and BRN3A, human pluripotent stem cells can be transcriptionally programmed to differentiate into a surprisingly uniform culture of cold- and mechano-sensing neurons. Although such a neuronal subtype is not found in mice, we identify molecular evidence for its existence in human sensory ganglia. Combining NGN2 and BRN3A programming with neural crest patterning, we produce two additional populations of sensory neurons, including a specialized touch receptor neuron subtype. Finally, we apply this system to model a rare inherited sensory disorder of touch and proprioception caused by inactivating mutations in PIEZO2. Together, these findings establish an approach to specify distinct sensory neuron subtypes in vitro, underscoring the utility of stem cell technology to capture human-specific features of physiology and disease., In Brief Nickolls et al. develop a method, using human stem cells, to generate specific types of sensory neurons that detect cold temperature and mechanical force. This approach uncovers a class of neuron found in humans, but not mice, and enables the modeling of a rare sensory disorder of touch and proprioception., Graphical Abstract

Acoustic signals are relayed from the ear to the brain via spiral ganglion neurons (SGNs) that receive auditory information from the cochlear inner hair cells (IHCs) and transmit that information to the cochlear nucleus of the brainstem. Physiologically distinct classes of SGNs have been characterized by their spontaneous firing rate and responses to sound and those physiological distinctions are thought to correspond to stereotyped synaptic positions on the IHC. More recently, single-cell profiling has identified multiple groups of SGNs based on transcriptional profiling; however, correlations between any of these groups and distinct neuronal physiology have not been determined. In this study, we show that expression of the POU (Pit-Oct-Unc) transcription factor Pou4f1 in type I SGNs in mice of both sexes correlates with a synaptic location on the modiolar side of IHCs. Conditional deletion of Pou4f1 in SGNs beginning in mice at embryonic day 13 rescues the early path-finding and apoptotic phenotypes reported for germline deletion of Pou4f1, resulting in a phenotypically normal development of SGN patterning. However, conditional deletion of Pou4f1 in SGNs alters the activation of Ca(2+) channels in IHCs primarily by increasing their voltage sensitivity. Moreover, the modiolar to pillar gradient of active zone Ca(2+) influx strength is eliminated. These results demonstrate that a subset of modiolar-targeted SGNs retain expression of Pou4f1 beyond the onset of hearing and suggest that this transcription factor plays an instructive role in presynaptic Ca(2+) signaling in IHCs. SIGNIFICANCE STATEMENT Physiologically distinct classes of type I spiral ganglion neurons (SGNs) are necessary to encode sound intensities spanning the audible range. Although anatomical studies have demonstrated structural correlates for some physiologically defined classes of type I SGNs, an understanding of the molecular pathways that specify each type is only now emerging. Here, we demonstrate that expression of the transcription factor Pou4f1 corresponds to a distinct subgroup of type I SGNs that synapse on the modiolar side of inner hair cells. The conditional deletion of Pou4f1 after SGN formation does not disrupt ganglion size or morphology, change the distribution of IHC synaptic locations, or affect the creation of synapses, but it does influence the voltage dependence and strength of Ca(2+) influx at presynaptic active zones in inner hair cells.

Age-related macular degeneration (AMD) is the foremost cause of blindness in people over the age of 60 worldwide. Clinically, this disease starts with distortion in central vision eventually leading to legal blindness. Vision loss has a significant impact on quality of life and incurs a substantial cost to the economy. Furthermore, AMD is a complex and progressive neurodegenerative disorder that triggers visual impairment due to the loss of retinal pigmented epithelium (RPE) and the light-sensitive photoreceptors that they support, protect and provide nutrition. Currently, there is no curative treatment for the most common form of this disease, i.e., dry AMD. A novel approach to treat AMD involves the transplantation of RPE cells derived from human induced pluripotent stem cells (iPSCs) in the outer retina. These iPSC-derived RPE cells not only show characteristics similar to native RPE but also could replace as well as regenerate damaged pathologic RPE and produce supportive growth factors and cytokines. Several clinical trials are being conducted taking advantage of a variety of cell- and tissue engineering-based approaches. Here, we present a simple, cost effective, and scalable cell-culture model for generation of purified RPE thus providing the foundation for developing an allogeneic cell therapy for AMD.

Background About 20–30 distinct Retinal Ganglion Cell (RGC) types transmit visual information from the retina to the brain. The developmental mechanisms by which RGCs are specified are still largely unknown. Brn3a is a member of the Brn3/Pou4f transcription factor family, which contains key regulators of RGC postmitotic specification. In particular, Brn3a ablation results in the loss of RGCs with small, thick and dense dendritic arbors (‘midget-like’ RGCs), and morphological changes in other RGC subpopulations. To identify downstream molecular mechanisms underlying Brn3a effects on RGC numbers and morphology, our group recently performed a RNA deep sequencing screen for Brn3a transcriptional targets in mouse RGCs and identified 180 candidate transcripts. Methods We now focus on a subset of 28 candidate genes encoding potential cell type determinant proteins. We validate and further define their retinal expression profile at five postnatal developmental time points between birth and adult stage, using in situ hybridization (ISH), RT-PCR and fluorescent immunodetection (IIF). Results We find that a majority of candidate genes are enriched in the ganglion cell layer during early stages of postnatal development, but dynamically change their expression profile. We also document transcript-specific expression differences for two example candidates, using RT-PCR and ISH. Brn3a dependency could be confirmed by ISH and IIF only for a fraction of our candidates. Conclusions Amongst our candidate Brn3a target genes, a majority demonstrated ganglion cell layer specificity, however only around two thirds showed Brn3a dependency. Some were previously implicated in RGC type specification, while others have known physiological functions in RGCs. Only three genes were found to be consistently regulated by Brn3a throughout postnatal retina development – Mapk10, Tusc5 and Cdh4. Electronic supplementary material The online version of this article (10.1186/s13064-018-0110-0) contains supplementary material, which is available to authorized users.

We studied reception of sex steroid hormones in the tissues of thyroid papillary cancer and benign tumor. Enhanced expression of AR and ERβ mRNA reflected malignant tumor growth. Nuclear factors Brn-3α and TRIM16 modulating expression of steroid hormones play an important role in the development of thyroid tumors. It was found that the level of TRIM16 mRNA is associated with the expression of ERβ, which seems to be mediated by its antiestrogen effect.

Background/Aims: Chronic Lymphocytic leukemia (CLL) is characterized by accumulation of cells in the G0/G1 phase of the cell cycle and resistance to apoptosis due to gene mutation or abnormal gene expression. In our previous study, constitutively photomorphogenic 1 (COP1) was shown to be upregulated in Binet C-phase CLL patients. Based on the negative regulation of COP1 in the repair of DNA damage, we further studied the function of COP1 in CLL cell apoptosis induced by fludarabine in vitro and in vivo. Methods: We analyzed the sensitivity of primary CLL cells to the fludarabine by CCK-8, and detected the expression of p53 in cells after drug treatment by western blot. Next, we constructed COP1 overexrpessing CLL cell line HG3, and analyzed the effect of COP1 overexpression on the HG3 cell’s apoptosis, and HG3 transplant mice survival with drug treatment. Results: Here, we found that primary CLL cells with high expression of COP1 showed low sensitivity to the drug and presented delayed enrichment of p53 protein than cells with low COP1 expressed. COP1 overexpression reduced HG3 cell sensitivity to the fludarabine treatment and inhibited cell apoptosis, and also retarded itself via autoubiquitination. The further study showed that COP1 promoted ubiquitin-dependent p53 degradation, which further disrupts the formation of the p53-Brn-3a complex and activation of Bcl-2 transcription. Moreover, mice engrafted with cells overexpressing COP1 showed a shortened survival, increased tumor cells burden in spleen and bone marrow (BM), and reduced tumor cell apoptosis even when fludarabine combined cyclophosphamide (F+C) therapy was administered. Conclusion: This study demonstrates that COP1 contributes to drug resistance of CLL cells to the fludarabine treatment in vitro and in vivo.

The habenula, located on the dorsal thalamic surface, is an emotional and reward processing center. As in the mammalian brain, the zebrafish habenula is divided into dorsal (dHb) and ventral (vHb) subdivisions that project to the interpeduncular nucleus and median raphe (MR) respectively. Previously, we have shown that kisspeptin 1 (Kiss1) expressing in the vHb, regulates the serotonin (5‐HT) system in the MR. However, the connectivity between the Kiss1 neurons and the 5‐HT system remains unknown. To resolve this issue, we generated a specific antibody against zebrafish Kiss1 receptor (Kiss‐R1); using this primary antibody we found intense immunohistochemical labeling in the ventro‐anterior corner of the MR (vaMR) but not in 5‐HT neurons, suggesting the potential involvement of interneurons in 5‐HT modulation by Kiss1. Double‐fluorescence labeling showed that the majority of habenular Kiss1 neurons are glutamatergic. In the MR region, Kiss1 fibers were mainly seen in close association with glutamatergic neurons and only scarcely within GABAergic and 5‐HT neurons. Our findings indicate that the habenular Kiss1 neurons potentially modulate the 5‐HT system primarily through glutamatergic neurotransmission via as yet uncharacterized interneurons. The neuropeptide kisspeptin (Kiss1) play a key role in vertebrate reproduction. We have previously shown modulatory role of habenular Kiss1 in the raphe serotonin (5‐HT) systems. This study proposed that the habenular Kiss1 neurons modulate the 5‐HT system primarily through glutamatergic neurotransmission, which provides an important insight for understanding of the modulation of 5‐HT system by the habenula‐raphe pathway.

The belly spot and tail (Bst+/−) mouse phenotype is caused by mutations of the ribosomal protein L24 (Rpl24). Among various phenotypes in Bst+/− mice, the most interesting are its retinal abnormalities, consisting of delayed closure of choroid fissures, decreased ganglion cells and subretinal vascularization. We further characterized the Bst+/− mouse and investigated the underlying molecular mechanisms to assess the feasibility of using this strain as a model for stem cell therapy of retinal degenerative diseases due to retinal ganglion cell (RGC) loss. We found that, although RGCs are significantly reduced in retinal ganglion cell layer in Bst+/− mouse, melanopsin+ RGCs, also called ipRGCs, appear to be unchanged. Pupillary light reflex was completely absent in Bst+/− mice but they had a normal circadian rhythm. In order to examine the pathological abnormalities in Bst+/− mice, we performed electron microscopy in RGC and found that mitochondria morphology was deformed, having irregular borders and lacking cristae. The complex activities of the mitochondrial electron transport chain were significantly decreased. Finally, for subretinal vascularization, we also found that angiogenesis is delayed in Bst+/− associated with delayed hyaloid regression. Characterization of Bst+/− retina suggests that the Bst+/− mouse strain could be a useful murine model. It might be used to explore further the pathogenesis and strategy of treatment of retinal degenerative diseases by employing stem cell technology., Summary: We characterized Bst+/− mice and found that pupillary light reflex was completely absent, which could be used as a readout for the efficacy of stem cell therapy in this model.

Glaucoma is a group of neurodegenerative diseases characterized by the progressive loss of retinal ganglion cells (RGCs) and their axons, and is the second leading cause of blindness worldwide. Elevated intraocular pressure is a well known risk factor for the development of glaucomatous optic neuropathy and pharmacological or surgical lowering of intraocular pressure represents a standard procedure in glaucoma treatment. However, the treatment options are limited and although lowering of intraocular pressure impedes disease progression, glaucoma cannot be cured by the currently available therapy concepts. In an acute short-term ocular hypertension model in rat, we characterize RGC loss, but also microglial cell activation and vascular alterations of the retina at certain time points. The combination of these three parameters might facilitate a better evaluation of the disease progression, and could further serve as a new model to test novel treatment strategies at certain time points. Acute ocular hypertension (OHT) was induced by the injection of magnetic microbeads into the rat anterior chamber angle (n = 22) with magnetic position control, leading to constant elevation of IOP. At certain time points post injection (4d, 7d, 10d, 14d and 21d), RGC loss, microglial activation, and microvascular pericyte (PC) coverage was analyzed using immunohistochemistry with corresponding specific markers (Brn3a, Iba1, NG2). Additionally, the tightness of the retinal vasculature was determined via injections of Texas Red labeled dextran (10 kDa) and subsequently analyzed for vascular leakage. For documentation, confocal laser-scanning microscopy was used, followed by cell counts, capillary length measurements and morphological and statistical analysis. The injection of magnetic microbeads led to a progressive loss of RGCs at the five time points investigated (20.07%, 29.52%, 41.80%, 61.40% and 76.57%). Microglial cells increased in number and displayed an activated morphology, as revealed by Iba1-positive cell number (150.23%, 175%, 429.25%,486.72% and 544.78%) and particle size analysis (205.49%, 203.37%, 412.84%, 333.37% and 299.77%) compared to contralateral control eyes. Pericyte coverage (NG2-positive PC/mm) displayed a significant reduction after 7d of OHT in central, and after 7d and 10d in peripheral retina. Despite these alterations, the tightness of the retinal vasculature remained unaltered at 14 and 21 days after OHT induction. While vascular tightness was unchanged in the course of OHT, a progressive loss of RGCs and activation of microglial cells was detected. Since a significant loss in RGCs was observed already at day 4 of experimental glaucoma, and since activated microglia peaked at day 10, we determined a time frame of 7–14 days after MB injection as potential optimum to study glaucoma mechanisms in this model.

Thalamocortical loops have been implicated in the control of higher-order cognitive functions, but advances in our understanding of the molecular underpinnings of neocortical organization have not been accompanied by similar analyses in the thalamus. Using expression-based correlation maps and the manual mapping of mouse and human datasets available in the Allen Brain Atlas, we identified a few individual regions and several sets of molecularly related nuclei that partially overlap with the classic grouping that is based on topographical localization and thalamocortical connections. These new molecular divisions of the adult thalamic complex are defined by the combinatorial expression of Tcf7l2, Lef1, Gbx2, Prox1, Pou4f1, Esrrg, and Six3 transcription factor genes. Further in silico and experimental analyses provided the evidence that TCF7L2 might be a pan-thalamic specifier. These results provide substantial insights into the “molecular logic” that underlies organization of the thalamic complex. Electronic supplementary material The online version of this article (doi:10.1007/s00429-015-1052-5) contains supplementary material, which is available to authorized users.

Generation of autoantigens of nuclear origin, like dsDNA and extractable nuclear antigens (ENA) have largely been associated with dysregulated apoptosis and defective clearance of apoptotic debris in SLE. Heat shock protein (HSP) 27 has been reported to have anti-apoptotic properties hence it was of interest to study the expression of HSP27 and its regulatory molecule Brn3a and hsa-miR-939 in SLE patients with distinct autoantibodies specificities. SLE patients were categorized into three subsets based on their distinct sero-positivity for either anti-dsDNA antibody alone (anti-dsDNA + group) or anti-ENA antibody alone (anti-ENA + group) or both (anti-dsDNA + ENA + group). We investigated the mRNA and protein expression of HSP27 and Brn3a in peripheral blood leukocytes (PBLs) by real-time reverse transcriptase PCR and Western blotting. Expression of apoptosis markers caspase 3 and poly (ADP-ribose) polymerase (PARP) was determined by Western blotting. Hsa-miR-939 expression was determined using TaqMan ® miRNA assay. In this study, we report significant downregulation of HSP27 in anti-ENA + patients and increased expression of caspase 3 and PARP in both anti-ENA + and anti-dsDNA + SLE subsets. A negative correlation was observed between the expression of HSP27 and apoptosis markers caspase 3 and PARP. Decreased Brn3a expression was observed in anti-ENA + SLE patients, which correlated positively with HSP27 expression. Expression of hsa-miR-939, which has a potential target site for Brn3a 3′ UTR, was also elevated specifically in anti-ENA + patients. The decreased expressions of HSP27, Brn3a along with elevated levels of hsa-miR-939 are selectively associated with anti-ENA + patients and HSP27 was observed to be inversely associated with apoptosis. These findings are suggestive of distinct regulatory processes operative in SLE patient subsets with different autoantibody specificities.

Glaucoma is a group of optic neuropathies characterized by the loss of retinal ganglion cells (RGCs). Since ocular hypertension (OHT) is a main risk factor, current therapies are predominantly based on lowering eye pressure. However, a subset of treated patients continues to lose vision. More research into pathological mechanisms underlying glaucoma is therefore warranted in order to develop novel therapeutic strategies. In this study we investigated the impact of OHT from eye to brain in mice.Monocular hypertension (mOHT) was induced in CD-1 mice by laser photocoagulation (LP) of the perilimbal and episcleral veins. The impact on the retina and its main direct target area, the superficial superior colliculus (sSC), was examined via immunostainings for Brn3a, VGluT2 and GFAP. Alterations in neuronal activity in V1 and extrastriate areas V2L and V2M were assessed using in situ hybridization for the activity reporter gene zif268.Transient mOHT resulted in diffuse and sectorial RGC degeneration. In the sSC contralateral to the OHT eye, a decrease in VGluT2 immunopositive synaptic connections was detected one week post LP, which appeared to be retinotopically linked to the sectorial RGC degeneration patterns. In parallel, hypoactivity was discerned in contralateral retinotopic projection zones in V1 and V2. Despite complete cortical reactivation 4 weeks post LP, in the sSC no evidence for recovery of RGC synapse density was found and also the concomitant inflammation was not completely resolved. Nevertheless, sSC neurons appeared healthy upon histological inspection and subsequent analysis of cell density revealed no differences between the ipsi- and contralateral sSC.In addition to RGC death, OHT induces loss of synaptic connections and neuronal activity in the visual pathway and is accompanied by an extensive immune response. Our findings stress the importance of looking beyond the eye and including the whole visual system in glaucoma research.

Optic nerve crush (ONC) and subsequent axonal damage can be used in rodents to study the mechanism of retinal ganglion cell (RGC) degeneration. Here, we examined electroretinograms (ERGs) in post-ONC mice to investigate changes in the positive scotopic threshold response (pSTR). We then compared these changes with molecular and morphological changes to identify early objective biomarkers of RGC dysfunction.Fifty 12-week-old C57BL/6 mice were included. ONC was used to induce axonal injury in the right eye of each animal, with the left eye used as a control. The expression of the RGC markers Brn3a and Brn3b was measured on days 1, 2, 3, 5 and 7 after ONC with quantitative real-time PCR. ERGs were recorded under dark adaptation with the stimulus intensity increasing from -6.2 to 0.43 log cd-s/m(2) on days 1, 2, 3, 5, 7 and 10 after ONC. The pSTR, a- and b-wave amplitudes were measured. Inner retinal thickness around the optic nerve head was measured with spectral-domain optical coherence tomography on days 0, 2, 5, 7 and 10 after ONC.The expression of Brn3a and Brn3b began to significantly decrease on day 1 and day 2, respectively (P 0.01). The amplitude of the pSTR underwent rapid, significant deterioration on day 3, after which it fell gradually (P 0.01), while the a- and b-wave amplitudes remained unchanged throughout the experiment. Inner retinal thickness gradually decreased, with the most significant reduction on day 10 (P 0.01).Decrease in pSTR likely reflected the early loss of RGC function after ONC and that declining expression of RGC-specific genes preceded anatomical and functional changes in the RGCs.

We report a case of a nine-year-old male who presented with facial nerve stimulation four years after cochlear implantation. Computed tomography was performed revealing a dilated internal auditory meatus and the cochlear implant electrode was found to be protruding into the fallopian canal at the level of the geniculate ganglion. Subsequent genetic analysis demonstrated X-linked deafness type 2 (DFNX2) caused by a novel c.769C > T nucleotide change in the POU domain, class 3, transcription factor 4 gene (POU3F4). Inactivation of electrodes 1 and 19-21 successfully abated facial nerve stimulation.

Significance While knowledge of signaling mechanisms orchestrating the development and diversification of peripheral somatosensory neurons is extensive, our understanding of the mechanisms controlling chemosensory neuron specification remains rudimentary. Lingually projecting sensory neurons of the geniculate ganglion are receptive to the five taste qualities, as well as temperature and tactile stimuli, but the mechanisms responsible for the diversification of the unique subpopulations that respond to one, or several, of these stimuli remain unknown. Here we demonstrate that the GDNF-Ret signaling pathway exerts a unique, dual function in peripheral taste system development and postnatal function. Ret acts embryonically to regulate the expression of the chemosensory master regulator Phox2b, thus inducing chemosensory differentiation, while postnatally acting to specify a molecularly unique subpopulation of lingual mechanoreceptors., The development of the taste system relies on the coordinated regulation of cues that direct the simultaneous development of both peripheral taste organs and innervating sensory ganglia, but the underlying mechanisms remain poorly understood. In this study, we describe a novel, biphasic function for glial cell line-derived neurotrophic factor (GDNF) in the development and subsequent diversification of chemosensory neurons within the geniculate ganglion (GG). GDNF, acting through the receptor tyrosine kinase Ret, regulates the expression of the chemosensory fate determinant Phox2b early in GG development. Ret−/− mice, but not Retfx/fx; Phox2b-Cre mice, display a profound loss of Phox2b expression with subsequent chemosensory innervation deficits, indicating that Ret is required for the initial amplification of Phox2b expression but not its maintenance. Ret expression is extinguished perinatally but reemerges postnatally in a subpopulation of large-diameter GG neurons expressing the mechanoreceptor marker NF200 and the GDNF coreceptor GFRα1. Intriguingly, we observed that ablation of these neurons in adult Ret-Cre/ERT2; Rosa26LSL-DTA mice caused a specific loss of tactile, but not chemical or thermal, electrophysiological responses. Overall, the GDNF-Ret pathway exerts two critical and distinct functions in the peripheral taste system: embryonic chemosensory cell fate determination and the specification of lingual mechanoreceptors.

In nocturnal rodents, voluntary wheel-running activity (WRA) represents a self-reinforcing behavior. We have previously demonstrated that WRA is markedly reduced in mice with a region-specific deletion of the transcription factor Pou4f1 (Brn3a), which leads to an ablation of the dorsal medial habenula (dMHb). The decrease in WRA in these dMHb-lesioned (dMHbCKO) mice suggests that the dMHb constitutes a critical center for conveying reinforcement by exercise. However, WRA also represents a prominent output of the circadian system, and the possibility remains that the dMHb is a source of input to the master circadian pacemaker located in the suprachiasmatic nucleus (SCN) of the hypothalamus. To test this hypothesis, we assessed the integrity of the circadian system in dMHbCKO mice. Here we show that the developmental lesion of the dMHb reduces WRA under both a light-dark cycle and constant darkness, increases the circadian period of WRA, but has no effect on the circadian amplitude or period of home cage activity or the daily amplitude of sleep stages, suggesting that the lengthening of period is a result of the decreased WRA in the mutant mice. Polysomnographic sleep recordings show that dMHbCKO mice have an overall unaltered daily amplitude of sleep stages but have fragmented sleep and an overall increase in total rapid eye movement (REM) sleep. Photoresponsiveness is intact in dMHbCKO mice, but compared with control animals, they reentrain faster to a 6-h abrupt phase delay protocol. Circadian changes in WRA of dMHbCKO mice do not appear to emerge within the central pacemaker, as circadian expression of the clock genes Per1 and Per2 within the SCN is normal. We do find some evidence for fragmented sleep and an overall increase in total REM sleep, supporting a model in which the dMHb is part of the neural circuitry encoding motivation and involved in the manifestation of some of the symptoms of depression.

Astrocytes are glial cells that support and protect neurons in the central nervous systems including the retina. Retinal ganglion cells (RGCs) are in contact with the astrocytes and our earlier findings showed the reduction of the number of cells in the ganglion cell layer in adult progranulin deficient mice. In the present study, we focused on the time of activation of the astrocytes and the alterations in the number of RGCs in the retina and optic nerve in progranulin deficient mice. Our findings showed that the number of Brn3a-positive cells was reduced and the expression of glial fibrillary acidic protein (GFAP) was increased in progranulin deficient mice. The progranulin deficient mice had a high expression of GFAP on postnatal day 9 (P9) but not on postnatal day 1. These mice also had a decrease in the number of the Brn3a-positive cells on P9. Taken together, these findings indicate that the absence of progranulin can affect the survival of RGCs subsequent the activation of astrocytes during retinal development.

Treatment of a mouse model of oxygen-induced retinopathy (OIR) with recombinant human Norrin (Norrie Disease Protein, gene: NDP) accelerates regrowth of the microvasculature into central ischemic regions of the neural retina, which are generated after treatment with 75% oxygen. While this reduces the average duration and severity of ischemia overall, we do not know if this accelerated recovery of the microvasculature results in any significant survival of retinal ganglion cells (RGCs). The purpose of this study was to investigate ganglion cell survival with and without the intravitreal injection of Norrin in the murine model of oxygen induced retinopathy (OIR), using two strains of mice: C57BL/6J and Thy1-YFP mice. Intravitreal injections of Norrin or vehicle were done after five days of exposure to 75% oxygen from ages P7 to P12. The C57BL/J mice were followed by Spectral-Domain Optical Coherence Tomography (SD-OCT), and the average nerve fiber layer (NFL) and inner-plexiform layer (IPL) thicknesses were measured at twenty-four locations per retina at P42. Additionally, some C57BL/J retinas were flat mounted and immunostained for the RGC marker, Brn3a, to compare the population density of surviving retinal ganglion cells. Using homozygous Thy1-YFP mice, single intrinsically fluorescent RGCs were imaged in live animals with a Micron-III imaging system at ages P21, 28 and P42. The relative percentage of YFP-fluorescent RGCs with dendritic arbors were compared. At age P42, the NFL was thicker in Norrin-injected OIR eyes, 14.4 μm, compared to Vehicle-injected OIR eyes, 13.3 μm (p = 0.01). In the superior retina, the average thickness of the IPL was greater in Norrin-injected OIR eyes, 37.7 μm, compared to Vehicle-injected OIR eyes, 34.6 μm (p = 0.04). Retinas from Norrin injected OIR mice had significantly more surviving RGCs (p = 0.03) than vehicle-injected mice. Based upon NFL thickness and counts of RGCs, we conclude that Norrin treatment, early in the ischemic phase, increased the relative population density of surviving RGCs in the central retinas of OIR mice.

The purpose of this work was to evaluate a potentially useful animal model, Meriones shawi (M.sh)-developing metabolic X syndrome, diabetes and possessing a visual streak similar to human macula-in the study of diabetic retinopathy and diabetic macular edema (DME). Type 2 diabetes (T2D) was induced by high fat diet administration in M.sh. Body weights, blood glucose levels were monitored throughout the study. Diabetic retinal histopathology was evaluated 3 and 7 months after diabetes induction. Retinal thickness was measured, retinal cell types were labeled by immunohistochemistry and the number of stained elements were quantified. Apoptosis was determined with TUNEL assay. T2D induced progressive changes in retinal histology. A significant decrease of retinal thickness and glial reactivity was observed without an increase in apoptosis rate. Photoreceptor outer segment degeneration was evident, with a significant decrease in the number of all cones and M-cone subtype, but-surprisingly-an increase in S-cones. Damage of the pigment epithelium was also confirmed. A decrease in the number and labeling intensity of parvalbumin- and calretinin-positive amacrine cells and a loss of ganglion cells was detected. Other cell types showed no evident alterations. No DME-like condition was noticed even after 7 months. M.sh could be a useful model to study the evolution of diabetic retinal pathology and to identify the role of hypertension and dyslipidemia in the development of the reported alterations. Longer follow up would be needed to evaluate the potential use of the visual streak in modeling human macular diseases.

The habenular complex in the epithalamus consists of distinct regions with diverse neuronal populations. Past studies have suggested a role for the habenula in voluntary exercise motivation and reinforcement of intracranial self-stimulation but have not assigned these effects to specific habenula subnuclei. Here, we have developed a genetic model in which neurons of the dorsal medial habenula (dMHb) are developmentally eliminated, via tissue-specific deletion of the transcription factorPou4f1(Brn3a). Mice with dMHb lesions perform poorly in motivation-based locomotor behaviors, such as voluntary wheel running and the accelerating rotarod, but show only minor abnormalities in gait and balance and exhibit normal levels of basal locomotion. These mice also show deficits in sucrose preference, but not in the forced swim test, two measures of depression-related phenotypes in rodents. We have also used Cre recombinase-mediated expression of channelrhodopsin-2 and halorhodopsin to activate dMHb neurons or silence their output in freely moving mice, respectively. Optical activation of the dMHbin vivosupports intracranial self-stimulation, showing that dMHb activity is intrinsically reinforcing, whereas optical silencing of dMHb outputs is aversive. Together, our findings demonstrate that the dMHb is involved in exercise motivation and the regulation of hedonic state, and is part of an intrinsic reinforcement circuit.

Aims To investigate the cause of retinal ganglion cell (RGC) loss in dystrophic aged Royal College of Surgeons (RCS) rats. Methods RCS-p+ (dystrophic) female rats of postnatal times (P365, P450 and P540) and age-matched RCS-p1 rdy+ (non-dystrophic) rats were used. In whole-mounted retinas, RGCs were doubly labelled with Fluorogold (FG) retrogradely transported from the superior colliculi and Brn3a immunohistochemistry. RGC axons were labelled with anti-neurofilament antibodies. Automatic image analysis techniques allowed quantification of the total population of RGCs per retina and construction of isodensity maps to investigate RGC topology. Results Dystrophic retinas showed at all times studied wedge-shaped sectors devoid of FG + and Brn3a + RGCs. These sectors were also devoid of neurofilament-labelled axons. The total number of FG + RGC and Brn3a + RGC per retina was significantly smaller in dystrophic rats at P540, revealing RGC death at this age. The total number of FG + RGCs was smaller than those of Brn3a + RGCs at P540, indicating a disturbance of the retrograde axonal transport at this age. Conclusions RGC double labelling documents that sectorial RGC loss in aged dystrophic RCS rats is mainly due to RGC death, although a deficit of the retrograde axonal transport exists also at the more advanced ages.

Much of the morbidity after traumatic brain injury (TBI) is associated with traumatic axonal injury (TAI). Although most TAI studies focus on corpus callosum white matter, the visual system has received increased interest. To assess visual system TAI, we developed a mouse model of optic nerve TAI. It is unknown, however, whether this TAI causes retinal ganglion cell (RGC) death. To address this issue, YFP (yellow fluorescent protein)-16 transgenic mice were subjected to mild TBI and followed from 2 to 28 days. Neither TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling)-positive or cleaved caspase-3-immunoreactive RGCs were observed from 2 to 28 days after TBI. Quantification of immunoreactivity of Brn3a, an RGC marker, demonstrated no RGC loss; parallel electron microscopic analysis confirmed RGC viability. Persistent RGC survival was also consistent with the finding of reorganization in the proximal axonal segments after TAI, wherein microglia/macrophages remained inactive. In contrast, activated microglia/macrophages closely enveloped the distal disconnected, degenerating axonal segments at 7 to 28 days after injury, thereby confirming that this model consistently evoked TAI followed by disconnection. Collectively, these data provide novel insight into the evolving pathobiology associated with TAI that will form a foundation for future studies exploring TAI therapy and its downstream consequences.

The Oct1 transcription factor is a potent regulator of stress responses, metabolism, and tumorigenicity. Although Oct1 is regulated by phosphorylation and ubiquitination, the presence and importance of other modifications is unknown. Here we show that Oct1 is modified by O-linked β-N-acetylglucosamine (O-GlcNAc) moieties. We map two sites of O-GlcNAcylation at positions T255 and S728 within human Oct1. Under anchorage-independent overgrowth conditions, Oct1 associates 3-fold more strongly with the Gadd45a promoter and mediates transcriptional repression. Increased binding correlates with quantitative reductions in Oct1 nuclear periphery-associated puncta, and a reduced association with lamin B1. The O-GlcNAc modification sites are important for both Gadd45a repression and anchorage-independent survival. In contrast to chronic overgrowth conditions, following acute nutrient starvation Oct1 mediates Gadd45a activation. The O-GlcNAc sites are also important for Gadd45a activation under these conditions. We also, for the first time, identify specific Oct1 ubiquitination sites. The findings suggest that Oct1 integrates metabolic and stress signals via O-GlcNAc modification to regulate target gene activity.—Kang, J., Shen, Z., Lim, J.-M., Handa, H., Wells, L., Tantin, D. Regulation of Oct1/Pou2f1 transcription activity by O-GlcNAcylation.

Pigment cells and neuronal cells both are derived from the neural crest. Here, we describe the Pit-Oct-Unc (POU) domain transcription factor Brn3a, normally involved in neuronal development, to be frequently expressed in melanoma, but not in melanocytes and nevi. RNAi-mediated silencing of Brn3a strongly reduced the viability of melanoma cell lines and decreased tumour growth in vivo. In melanoma cell lines, inhibition of Brn3a caused DNA double-strand breaks as evidenced by Mre11/Rad50-containing nuclear foci. Activated DNA damage signalling caused stabilization of the tumour suppressor p53, which resulted in cell cycle arrest and apoptosis. When Brn3a was ectopically expressed in primary melanocytes and fibroblasts, anchorage-independent growth was increased. In tumourigenic melanocytes and fibroblasts, Brn3a accelerated tumour growth in vivo. Furthermore, Brn3a cooperated with proliferation pathways such as oncogenic BRAF, by reducing oncogene-induced senescence in non-malignant melanocytes. Together, these results identify Brn3a as a new factor in melanoma that is essential for melanoma cell survival and that promotes melanocytic transformation and tumourigenesis.

In mammals, the sensory hair cells and auditory neurons do not spontaneously regenerate and their loss results in permanent hearing impairment. Stem cell therapy is one emerging strategy that is being investigated to overcome the loss of sensory cells after hearing loss. To successfully replace auditory neurons, stem cell-derived neurons must be electrically active, capable of organized outgrowth of processes, and of making functional connections with appropriate tissues. We have developed an in vitro assay to test these parameters using cocultures of developing cochlear explants together with neural progenitors derived from human embryonic stem cells (hESCs). We found that these neural progenitors are electrically active and extend their neurites toward the sensory hair cells in cochlear explants. Importantly, this neurite extension was found to be significantly greater when neural progenitors were predifferentiated toward a neural crest-like lineage. When grown in coculture with hair cells only (denervated cochlear explants), stem cell-derived processes were capable of locating and growing along the hair cell rows in an en passant-like manner. Many presynaptic terminals (synapsin 1-positive) were observed between hair cells and stem cell-derived processes in vitro. These results suggest that differentiated hESC-derived neural progenitors may be useful for developing therapies directed at auditory nerve replacement, including complementing emerging hair cell regeneration therapies.

Intrinsically photosensitive retinal ganglion cells (ipRGCs) respond directly to light and are responsible of the synchronization of the circadian rhythm with the photic stimulus and for the pupillary light reflex. To quantify the total population of rat-ipRGCs and to assess their spatial distribution we have developed an automated routine and used neighbour maps. Moreover, in all analysed retinas we have studied the general population of RGCs – identified by their Brn3a expression – and the population of ipRGCs – identified by melanopsin immunodetection – thus allowing the co-analysis of their topography. Our results show that the total mean number ± standard deviation of ipRGCs in the albino rat is 2047 ± 309. Their distribution in the retina seems to be complementary to that of Brn3a + RGCs, being denser in the periphery, especially in the superior retina where their highest densities are found in the temporal quadrant, above the visual streak. In addition, by tracing the retinas from both superior colliculi, we have also determined that 90.62% of the ipRGC project to these central targets.

Purpose To study the effect of topical administration of bromfenac, a nonsteroidal anti-inflammatory drug (NSAID), on retinal gliosis and levels of prostaglandin E2 (PGE2) after complete optic nerve crush (ONC). Methods Adult albino rats were divided into the following groups (n = 8 retinas/group): (1) intact, (2) intact and bromfenac treatment (twice a day during 7 days), (3) ONC (7 days), and (4) ONC (7 days) + bromfenac treatment (twice a day during 7 days). Animals from groups 3 and 4 were imaged in vivo with spectral-domain optical coherence tomography (SD-OCT) before the procedure and 15 minutes, 3, 5, or 7 days later. Retinas from all groups were analyzed by immunodetection, Western blotting, or enzyme-linked immunoabsorbent assay (ELISA). Results Quantification of Brn3a (brain-specific homeobox/POU domain protein 3A) +RGCs (retinal ganglion cells) in cross sections showed that bromfenac treatment does not accelerate ONC-induced degeneration. Cellular retinaldehyde binding protein 1 regulation indicated that bromfenac improves retinal homeostasis in injured retinas. Spectral-domain OCT showed that the thickness of the retina and the retinal nerve fiber layer at 7 days post ONC was significantly reduced in bromfenac-treated animals when compared to untreated animals. In agreement with these data, hypertrophy of astrocytes and Muller cells and expression of glial fibrillary acidic protein and vimentin were greatly diminished by bromfenac treatment. While no changes in cyclooxygenase (COX) enzyme COX1 and COX2 expression were observed, there was a significant increase of PGE2 after ONC that was controlled by bromfenac treatment. Conclusions Topical administration of bromfenac is an efficient and noninvasive treatment to control the retinal gliosis and release of proinflammatory mediators that follow a massive insult to the RGC population.

The Brn-3a POU transcription factor is associated with survival and the differentiation of sensory neuronal cells during development. Brn-3a mediates its effects either by the direct regulation of target genes or indirectly upon interaction with proteins such as p53. Brn-3a differentially regulates p53-mediated gene expression and modifies its effect on cell fate. Here we show that, like Bax, Brn-3a antagonizes p53-mediated transcription of another proapoptotic target, Noxa, significantly reducing transactivation of the Noxa promoter by p53. This effect requires the p53 binding site, and electrophoretic mobility shift assay studies suggest that Brn-3a is associated with p53 when it is bound to its site in the Noxa promoter. The wild type but not the mutant promoter can be immunoprecipitated with Brn-3a in chromatin immunoprecipitation assays. Thus, Brn-3a may act by preventing the recruitment of cofactors required for p53 to transactivate this promoter. The co-expression of Brn-3a and p53 results in decreased endogenous Noxa protein in the neuronal cell line, ND7, suggesting a direct functional effect of this interaction. Moreover, there is a significant elevation of both proapoptotic Bax and Noxa proteins in sensory neuronal tissue taken from Brn-3a-/- embryos during development, compared with wild type controls. Striking changes occurred at embryonic day 14.5, a time that precedes a significant loss of specific neurons in the mutant embryos, but not at embryonic day 16.5 when Brn-3a-expressing cells are already lost by apoptosis. Therefore, the lack of antagonism by Brn-3a on activation of proapoptotic p53 target genes may contribute to the increased apoptosis seen in the Brn-3a-/- embryos. These results support a crucial role for Brn-3a in determining the pathway taken by p53 when co-expressed during development and thus in controlling the fate of these cells.

Animal models have been developed to investigate aspects of stress, anxiety, and depression, but our understanding of the circuitry underlying these models remains incomplete. Prior studies of the habenula, a poorly understood nucleus in the dorsal diencephalon, suggest that projections to the medial habenula (MHb) regulate fear and anxiety responses, whereas the lateral habenula (LHb) is involved in the expression of learned helplessness, a model of depression. Tissue-specific deletion of the transcription factor Pou4f1 in the dorsal MHb (dMHb) results in a developmental lesion of this subnucleus. These dMHb-ablated mice show deficits in voluntary exercise, a possible correlate of depression. Here we explore the role of the dMHb in mood-related behaviors and intrinsic reinforcement. Lesions of the dMHb do not elicit changes in contextual conditioned fear. However, dMHb-lesioned mice exhibit shorter immobility time in the tail suspension test, another model of depression. dMHb-lesioned mice also display increased vulnerability to the induction of learned helplessness. However, this effect is not due specifically to the dMHb lesion, but appears to result fromPou4f1haploinsufficiency elsewhere in the nervous system.Pou4f1haploinsufficiency does not produce the other phenotypes associated with dMHb lesions. Using optogenetic intracranial self-stimulation, intrinsic reinforcement by the dMHb can be mapped to a specific population of neurokinin-expressing habenula neurons. Together, our data show that the dMHb is involved in the regulation of multiple mood-related behaviors, but also support the idea that these behaviors do not reflect a single functional pathway.

We have studied the effect of α2-adrenergic receptor stimulation on the total excitotoxically injured chicken retinal ganglion cell population. N-methyl-D-aspartate (NMDA) was intraocularly injected at embryonic day 18 and Brn3a positive retinal ganglion cells (Brn3a+ RGCs) were counted in flat-mounted retinas using automated routines. The number and distribution of the Brn3a+ RGCs were analyzed in series of normal retinas from embryonic day 8 to post-hatch day 11 retinas and in retinas 7 or 14 days post NMDA lesion. The total number of Brn3a+ RGCs in the post-hatch retina was approximately 1.9x106 with a density of approximately 9.2x103 cells/mm2. The isodensity maps of normal retina showed that the density decreased with age as the retinal size increased. In contrast to previous studies, we did not find any specific region with increased RGC density, rather the Brn3a+ RGCs were homogeneously distributed over the central retina with decreasing density in the periphery and in the region of the pecten oculli. Injection of 5-10 μg NMDA caused 30-50% loss of Brn3a+ cells and the loss was more severe in the dorsal than in the ventral retina. Pretreatment with brimonidine reduced the loss of Brn3a+ cells both 7 and 14 days post lesion and the protective effect was higher in the dorsal than in the ventral retina. We conclude that α2-adrenergic receptor stimulation reduced the impact of the excitotoxic injury in chicken similarly to what has been shown in mammals. Furthermore, the data show that the RGCs are evenly distributed over in the retina, which challenges previous results that indicate the presence of specific high RGC-density regions of the chicken retina.

Glaucomatous optic neuropathies are characterized by progressive loss of retinal ganglion cells (RGCs), the neurons that connect the eye to the brain. Quantification of these RGCs is a cornerstone in experimental optic neuropathy research and commonly performed via manually quantifying parts of the retina. However, this is a time-consuming process subject to inter- and intra-observer variability. Here we present a freely available ImageJ script to semi-automatically quantify RGCs in entire retinal flatmounts after immunostaining for the RGC-specific transcription factor Brn3a. The blob-like signal of Brn3a-immunopositive RGCs is enhanced via eigenvalues of the Hessian matrix and the resulting local maxima are counted as RGCs. After the user has outlined the retinal flatmount area, the total RGC number and retinal area are reported and an isodensity map, showing the RGC density distribution across the retina, is created. The semi-automated quantification shows a very strong correlation (Pearson's r ≥ 0.99) with manual counts for both widefield and confocal images, thereby validating the data generated via the developed script. Moreover, application of this method in established glaucomatous optic neuropathy models such as N-methyl-D-aspartate-induced excitotoxicity, optic nerve crush and laser-induced ocular hypertension revealed RGC loss conform with literature. Compared to manual counting, the described automated quantification method is faster and shows user-independent consistency. Furthermore, as the script detects the RGC number in entire retinal flatmounts, the method allows detection of regional differences in RGC density. As such, it can help advance research investigating the degenerative mechanisms of glaucomatous optic neuropathies and the effectiveness of new neuroprotective treatments. Because the script is flexible and easy to optimize due to a low number of critical parameters, it can potentially be applied in combination with other tissues or alternative labeling protocols.

Highlights • The circadian behaviour of two models of lysosomal storage disorders were examined. • Npc1 mutant mice show no core circadian defects. • Hexb knockouts show potential circadian activity disturbances. • Contrasting pathology in LSDs may differentially contribute to circadian regulation., Sleep and circadian rhythm disruption is frequently associated with neurodegenerative disease, yet it is unclear how the specific pathology in these disorders leads to abnormal rest/activity profiles. To investigate whether the pathological features of lysosomal storage disorders (LSDs) influence the core molecular clock or the circadian behavioural abnormalities reported in some patients, we examined mouse models of Niemann-Pick Type-C (Npc1 mutant, Npc1nih) and Sandhoff (Hexb knockout, Hexb−/−) disease using wheel-running activity measurement, neuropathology and clock gene expression analysis. Both mutants exhibited regular, entrained rest/activity patterns under light:dark (LD) conditions despite the onset of their respective neurodegenerative phenotypes. A slightly shortened free-running period and changes in Per1 gene expression were observed in Hexb−/− mice under constant dark conditions (DD); however, no overt neuropathology was detected in the suprachiasmatic nucleus (SCN). Conversely, despite extensive cholesterol accumulation in the SCN of Npc1nih mutants, no circadian disruption was observed under constant conditions. Our results indicate the accumulation of specific metabolites in LSDs may differentially contribute to circadian deregulation at the molecular and behavioural level.

MicroRNAs have emerged as key posttranscriptional regulators of gene expression during vertebrate development. We show that themiR-200family plays a crucial role for the proper generation and survival of ventral neuronal populations in the murine midbrain/hindbrain region, including midbrain dopaminergic neurons, by directly targeting the pluripotency factor Sox2 and the cell-cycle regulator E2F3 in neural stem/progenitor cells. The lack of a negative regulation of Sox2 and E2F3 bymiR-200in conditionalDicer1mutants (En1+/Cre;Dicer1flox/floxmice) and aftermiR-200knockdownin vitroleads to a strongly reduced cell-cycle exit and neuronal differentiation of ventral midbrain/hindbrain (vMH) neural progenitors, whereas the opposite effect is seen aftermiR-200overexpression in primary vMH cells. Expression ofmiR-200is in turn directly regulated by Sox2 and E2F3, thereby establishing a unilateral negative feedback loop required for the cell-cycle exit and neuronal differentiation of neural stem/progenitor cells. Our findings suggest that the posttranscriptional regulation of Sox2 and E2F3 bymiR-200family members might be a general mechanism to control the transition from a pluripotent/multipotent stem/progenitor cell to a postmitotic and more differentiated cell.

The sensory neurons of the dorsal root ganglia (DRG) must project accurately to their central targets to convey proprioceptive, nociceptive and mechanoreceptive information to the spinal cord. How these different sensory modalities and central connectivities are specified and coordinated still remains unclear. Given the expression of the POU homeodomain transcription factors Brn3a/Pou4f1 and Brn3b/Pou4f2 in DRG and spinal cord sensory neurons, we determined the subtype specification of DRG and spinal cord sensory neurons as well as DRG central projections in Brn3a and Brn3b single and double mutant mice. Inactivation of either or both genes causes no gross abnormalities in early spinal cord neurogenesis; however, in Brn3a single and Brn3a;Brn3b double mutant mice, sensory afferent axons from the DRG fail to form normal trajectories in the spinal cord. The TrkA(+) afferents remain outside the dorsal horn and fail to extend into the spinal cord, while the projections of TrkC(+) proprioceptive afferents into the ventral horn are also impaired. Moreover, Brn3a mutant DRGs are defective in sensory neuron specification, as marked by the excessive generation of TrkB(+) and TrkC(+) neurons as well as TrkA(+)/TrkB(+) and TrkA(+)/TrkC(+) double positive cells at early embryonic stages. At later stages in the mutant, TrkB(+), TrkC(+) and parvalbumin(+) neurons diminish while there is a significant increase of CGRP(+) and c-ret(+) neurons. In addition, Brn3a mutant DRGs display a dramatic down-regulation of Runx1 expression, suggesting that the regulation of DRG sensory neuron specification by Brn3a is mediated in part by Runx1. Our results together demonstrate a critical role for Brn3a in generating DRG sensory neuron diversity and regulating sensory afferent projections to the central targets.

Several subtypes of melanopsin-expressing, intrinsically photosensitive retinal ganglion cells (ipRGCs) have been reported. The M1 type of ipRGCs exhibit distinct properties compared with the remaining (non-M1) cells. They differ not only in their soma size and dendritic arbor, but also in their physiological properties, projection patterns, and functions. However, it is not known how these differences arise. We tested the hypothesis that M1 and non-M1 cells express Brn3 transcription factors differentially. The Brn3 family of class IV POU-domain transcription factors (Brn3a, Brn3b, and Brn3c) is involved in the regulation of differentiation, dendritic stratification, and axonal projection of retinal ganglion cells during development. By using double immunofluorescence for Brn3 transcription factors and melanopsin, and with elaborate morphometric analyses, we show in mouse retina that neither Brn3a nor Brn3c are expressed in ipRGCs. However, Brn3b is expressed in a subset of ipRGCs, particularly those with larger somas and lower melanopsin levels, suggesting that Brn3b is expressed preferentially in the non-M1 cells. By using dendritic stratification to distinguish M1 from non-M1 cells, we found that whereas nearly all non-M1 cells expressed Brn3b, a vast majority of the M1 cells were negative for Brn3b. Interestingly, in the small proportion of the M1 cells that did express Brn3b, the expression level of Brn3b was significantly lower than in the non-M1 cells. These results provide insights about how expression of specific molecules in a ganglion cell could be linked to its role in visual function.

The presence of a widespread endocannabinoid (eCB) system within the nervous system, including the retina, has been demonstrated in recent years. Expression patterns of the cannabinoid receptor type 1 (CB1R) and enzyme fatty acid amide hydrolase (FAAH) are available for rodents, but data for humans and monkeys are scarce. We, therefore, thoroughly examined the distribution pattern of CB1R and FAAH throughout the retina of the vervet monkey (Chlorocebus sabeus) using confocal microscopy. Our results demonstrate that CB1R and FAAH are expressed throughout the retina, from the foveal pit to the far periphery. CB1R and FAAH are present in the photoreceptor, outer plexiform, inner nuclear, inner plexiform, and retinal ganglion cell layers (PRL, OPL, INL, IPL, and RGCL, respectively). More specifically, in PRL, CB1R and FAAH are preferentially expressed in cones of the central retina. In OPL, these two components of the eCB system are concentrated not only in the cone pedicles but also in rod spherules with, however, a less intense staining pattern. Triple-labeling immunofluorescence revealed that both cone and rod bipolar cells express CB1R and FAAH. Heavy staining is detected in RGC somas and axons. Neither CB1R nor FAAH are found in the retinal glia, the Müller cells. These data indicate that the eCB system is present throughout the primate retina and is ideally positioned to modulate central and peripheral retinal functions.

Purpose Intrinsically photosensitive retinal ganglion cells (ipRGCs) express the photopigment melanopsin (OPN4) and are mainly responsible for non-image-forming visual tasks such as circadian photoentrainment and the pupillary light reflex. Compared to other classes of RGCs, ipRGCs are more resistant to cell death in several experimental models such as ocular hypertension, optic nerve transection, and others. Here, we tested whether ipRGCs are also resistant to N-methyl-D-aspartic acid (NMDA)-induced excitotoxicity. Methods Mice were injected intravitreally with NMDA, and subsequent expression levels of Opn4 and Brn3a mRNA were analyzed with semiquantitative real-time PCR. Cells immunopositive for BRN3A and OPN4 were quantified in retinal flat mounts of NMDA- and PBS-injected eyes. The molecular response of the retina to NMDA treatment was analyzed with real-time PCR and western blotting. Intravitreal injections of wortmannin and AG-490 were used to inhibit phosphatidylinositol 3-kinase (PI3K)/AKT and Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling, respectively. Results In contrast to retinal Brn3a expression and BRN3A-containing cells, levels of Opn4 mRNA and the number of OPN4-expressing cells were not reduced after NMDA injection. Survival of ipRGCs after NMDA injection was not strain specific, did not require the presence of photoreceptor cells, and did not depend on PI3K/AKT or JAK/STAT signaling, although both signaling pathways were activated after NMDA treatment. Conclusions Our data support the existence of an efficient survival system for ipRGCs. This system does not depend on PI3K/AKT or JAK/STAT signaling. Identification of the responsible molecular survival mechanisms may provide clues to protect “traditional” ganglion cells in diseases such as glaucoma.

The host cellular transcription factor, BRN3A, has been observed to play a vital role in cancer of the uterine cervix. BRN3A possesses multipartite functions, which include transcription of the genes of the high-risk HPVs and mediation of cellular changes in the host. In this study, we made an effort to decipher the regulation of BRN3A in cervical cancer cells by studying its interaction with different components of the cell.In cervical cancer cells, the endogenous HIPK2 was induced through cisplatin treatment, and then, its subsequent effect on BRN3A was primarily investigated through co-immunostaining and western blotting as HIPK2 has been observed to act as a co-repressor of Brn3a. The physical interaction of the two proteins was analyzed through co-immunoprecipitation. We resorted to chromatin immunoprecipitation in order to testify the autoregulatory pathway of BRN3A in cervical cancer cells. Interaction of BRN3A with cellular components, p73 and active form of JNK, was also studied through co-immunostaining.We observed that BRN3A is independent of the regulative activity of HIPK2 and undergoes positive autoregulation in cervical cancer cells. Interestingly, during the study, it was revealed that BRN3A is unaffected by the treatment of cisplatin. Interaction of BRN3A with p73 and phosphorylated JNK in cervical cancer cells, observed in the present study, would help in understanding the molecular mechanism directed by BRN3A.BRN3A possesses anti-apoptotic property, and considering the above results, it may be regarded as the key component in promoting tumorigenic growth in the uterine cervical cells.

LIM homeodomain transcription factors, Lmx1a and Lmx1b, are required for the development of midbrain dopaminergic (mDA) neurons. Lmx1b is required for the specification and maintenance of mDA neurons, primarily due to its role in isthmic organizer development that is essential for the induction of mDA neurons. Here, we conditionally deleted Lmx1b in the ventral neural tube usingShhCreand found thatLmx1bconditional mutant mouse embryos show no defect in the development and maintenance of mDA neurons. In addition,Dreher(Lmx1a mutant) embryos display only a moderate reduction in the number of mDA neurons, suggesting that the related family member Lmx1b might compensate for Lmx1a function. We therefore generatedLmx1aandLmx1bdouble mutants. Severe loss of mDA neurons occurred inLmx1adr/dr;ShhCre/+;Lmx1bf/fdouble mutants due to essential roles for Lmx1a and Lmx1b in regulating the proliferation and neuronal commitment of mDA progenitors through the expression of Wnt1 and Ngn2, respectively. Lmx1a and Lmx1b also negatively regulateHes1expression and consequently cell cycle exit through activation of p27Kip1expression. In addition, Lmx1a and Lmx1b also regulate the expression of floor plate genes such asCorinandSlit2and specification of postmitotic mDA neurons. These defects were more severe with decreasing gene dosage of Lmx1a and Lmx1b or observed only when all four copies of Lmx1a and Lmx1b genes were inactivated. Together, our results demonstrate that Lmx1a and Lmx1b function cooperatively to regulate proliferation, specification, and differentiation of mDA progenitors, including their floor plate-like properties.

The combinatorial expression of transcription factors frequently marks cellular identity in the nervous system, yet how these factors interact to determine specific neuronal phenotypes is not well understood. Sensory neurons of the trigeminal ganglion (TG) and dorsal root ganglia (DRG) coexpress the homeodomain transcription factors Brn3a and Islet1, and past work has revealed partially overlapping programs of gene expression downstream of these factors. Here we examine sensory development in Brn3a/Islet1 double knock-out (DKO) mice. Sensory neurogenesis and the formation of the TG and DRG occur in DKO embryos, but the DRG are dorsally displaced, and the peripheral projections of the ganglia are markedly disturbed. Sensory neurons in DKO embryos show a profound loss of all early markers of sensory subtypes, including the Ntrk neurotrophin receptors, and the runt-family transcription factors Runx1 and Runx3. Examination of global gene expression in the E12.5 DRG of single and double mutant embryos shows that Brn3a and Islet1 are together required for nearly all aspects of sensory-specific gene expression, including several newly identified sensory markers. On a majority of targets, Brn3a and Islet1 exhibit negative epistasis, in which the effects of the individual knock-out alleles are less than additive in the DKO. Smaller subsets of targets exhibit positive epistasis, or are regulated exclusively by one factor. Brn3a/Islet1 double mutants also fail to developmentally repress neurogenic bHLH genes, and in vivo chromatin immunoprecipitation shows that Islet1 binds to a known Brn3a-regulated enhancer in the neurod4 gene, suggesting a mechanism of interaction between these genes.

Retinal ganglion cells (RGCs) are the projection neurons from the eye to the brain and their loss results in visual impairment in a number of diseases. Transcription factors with a homeodomain can translocate between cells and, in at least one reported case, can stimulate neuronal survival. Otx2 is a homeoprotein transcription factorexpressed in the retina that is taken up by RGCs. We thus hypothesized that Otx2 capture could regulate the survival of adult RGCs. We report that Otx2 stimulates the survival of adult mouse and rat RGCsin vitroand protects RGCs against NMDA-induced toxicityin vivoin mice. In the latter model, Otx2 also preserves visual acuity.

SummaryIt is a long-standing question how developing motor and sensory neuron projections cooperatively form a common principal grid of peripheral nerve pathways relaying behavioral outputs and somatosensory inputs. Here, we explored this issue through targeted cell lineage and gene manipulation in mouse, combined with in vitro live axon imaging. In the absence of motor projections, dorsal (epaxial) and ventral (hypaxial) sensory projections form in a randomized manner, while removal of EphA3/4 receptor tyrosine kinases expressed by epaxial motor axons triggers selective failure to form epaxial sensory projections. EphA3/4 act non-cell-autonomously by inducing sensory axons to track along preformed epaxial motor projections. This involves cognate ephrin-A proteins on sensory axons but is independent from EphA3/4 signaling in motor axons proper. Assembly of peripheral nerve pathways thus involves motor axon subtype-specific signals that couple sensory projections to discrete motor pathways.

8 p., 5 figures and references., The components of the molecular codes needed to specify the different neuronal populations present in the basal neural tube are being identified. These codes become more intricate as we move to more anterior regions of the central nervous system. The aim of this study is to thoroughly analyze the expression pattern of Nkx6.1, Nkx6.2, and Pou4f1. These three genes are candidates to play an important role in the determination and differentiation of the basal nuclei of the mesencephalon and diencephalon. The results obtained have shown that there is a longitudinal domain positive for both Nkx6.1 and Nkx6.2 that is medial to the Pou4f1-positive red nucleus. This domain could correspond to part of the reticular formation, which extends from the diencephalon and the mesencephalon. The nuclei integrated in this domain would be the rostral interstitial nucleus, the interstitial nucleus of Cajal, and a mesencephalic equivalent to these nuclei., Grant sponsor: MICINN; Grant numbers: BFU2008-03708/BFI, BFU2008-00588, CSD2007-00023; Grant sponsor: TV3 Marato; Grant number: 062232; Grant sponsor: European Commission; Grant number: LSHG-CT-2004-512003.

The t(8;21)(q22;q22) translocation, present in approximately 5% of adult acute myeloid leukemia (AML) cases, produces the AML1/ETO (AE) fusion protein. Dysregulation of the Pit/Oct/Unc (POU) domain-containing transcription factor POU4F1 is a recurring abnormality in t(8;21) AML. In this study, we showed that POU4F1 overexpression is highly correlated with, but not caused by, AE. We observed that AE markedly increases the self-renewal capacity of myeloid progenitors from murine bone marrow or fetal liver and drives the expansion of these cells in liquid culture. POU4F1 is neither necessary nor sufficient for these AE-dependent properties, suggesting that it contributes to leukemia through novel mechanisms. To identify targets of POU4F1, we performed gene expression profiling in primary mouse cells with genetically defined levels of POU4F1 and identified 140 differentially expressed genes. This expression signature was significantly enriched in human t(8;21) AML samples and was sufficient to cluster t(8;21) AML samples in an unsupervised hierarchical analysis. Among the most highly differentially expressed genes, half are known AML1/ETO targets, implying that the unique transcriptional signature of t(8;21) AML is, in part, attributable to POU4F1 and not AML1/ETO itself. These genes provide novel candidates for understanding the biology and developing therapeutic approaches for t(8;21) AML.

Ewing's sarcoma family tumors (ESFTs or EFTs) express neuronal markers, which indicates they may originate from cells at least partly committed to neuronal lineage. However, recent publications suggest EFT originates in mesenchymal stem cells, and EWS/ETS fusion proteins characteristic of EFT activate neuronal marker expression to confer a neural phenotype on EFT. Here we show that the neuronal marker BRN3A/POU4F1 is expressed abundantly at the protein level in primary EFT but not in rhabdomyosarcoma and neuroblastoma, and EFT cells exhibit high activity of the BRN3A proximal autoregulatory region. EWS/FLI-1 siRNA reduces BRN3A expression and promoter activity and EWS/ETS proteins are bound to the BRN3A locus, suggesting a direct function for EWS/ETS proteins in control of BRN3A expression. Differentiation-associated and autoregulatory activities of BRN3A are respectively impaired and altered in EFT cells, and EWS/FLI-1 siRNA can restore some BRN3A function. A potentially novel function for BRN3A in EFT cells is identified. These results extend the hypothesis that EWS/ETS proteins induce expression of neuronal markers such as BRN3A in EFT by showing that the function of those same markers may be restricted or controlled in an EWS/ETS-dependent manner.

Pituitary adenylate cyclase activating polypeptide (PACAP) and its high affinity receptor PAC1 are expressed in mammalian retina and involved in processing light information. However, their roles during retinogenesis remain largely elusive. Previously, we have generated transgenic mice overexpressing the human PAC1 receptor, and shown that PACAP signaling is essential for normal development of the central nervous system. In this study, we show for the first time that PACAP signaling plays an important role in the development of retina, particularly in the genesis of GABAergic amacrine cells. Overexpression of the PAC1 receptor leads to an early exit from retinal proliferation, reduced production of GABAergic neurons, and a marked decline in visual function. These data demonstrate that an appropriate level of PACAP signaling is required for normal retinogenesis and visual function. This finding may have implications in GABAergic neuron-related neurological conditions.

The POU-domain transcription factor Brn3a is expressed in developing sensory neurons at all levels of the neural axis, including the trigeminal ganglion, hindbrain sensory ganglia, and dorsal root ganglia. Changes in global gene expression in the trigeminal ganglion from E11.5 to E13.5 reflect the repression of early neurogenic genes, exit from the cell cycle, and initiation of the expression of definitive markers of sensory function. A majority of these developmental changes are perturbed in the trigeminal ganglia of Brn3a knockout mice. At E13.5, Brn3a(-/-) trigeminal neurons fail to repress a battery of developmental regulators that are highly expressed at E11.5 and are normally down-regulated as development progresses, and also fail to appropriately activate a set of definitive sensory genes. Remarkably, developing Brn3a(-/-) trigeminal neurons also ectopically express multiple regulatory genes associated with cardiac and/or cranial mesoderm development, although definitive myogenic programs are not activated. The majority of these genes are not ectopically expressed in the dorsal root ganglia of Brn3a null mice, perhaps due to redundant mechanisms of repression at spinal levels. These results underscore the importance of gene repression in regulating neuronal development, and the need for unbiased screens in the determination of developmental gene regulatory programs.

The neurons in the ventral mesencephalon (VM) are organized into several nuclei consisting of distinct neuronal populations. These include the dopaminergic (DA) neurons of the substania nigra and ventral tegmental area, the oculomotor (OM) neurons that innervate the muscles controlling eye movement, and the reticular neurons of the red nucleus (RN) involved in motor control and coordination reviewed in Puelles (2007). The factors and genes that control the differentiation of the various neuronal populations in the VM have been extensively studied in the mouse and other model organisms but little is known about the progenitors and their protein expression in the developing human brain. In this study we analyze if key regulators identified in rodents are also expressed in the human VM during embryonic development. We report that BLBP and LMX1A mark the floor plate and that FOXA2 is expressed in both the floor plate and basal plate of the human VM. The proneural transcription factors NGN2 and MASH1 are expressed in the ventricular zone of the human VM within and lateral to the floor plate. The post-mitotic DA neurons express TH as well as NURR1 and PITX3. ISL1 and BRN3A can be used to detect the cells of OM and RN, respectively. We show that many key developmental control factors are expressed in a temporal and spatial manner in the human VM essentially corresponding to what has been observed in the mouse. This data therefore suggest similar roles for these factors also in human VM development and dopamine neurogenesis.