Remote Ischemic Post-Conditioning Therapy is Protective in Mouse Model of Traumatic Optic Neuropathy.

Traumatic optic neuropathy (TON) is characterized by visual dysfunction after indirect or direct injury to the optic nerve following blunt head trauma. TON is associated with increased oxidative stress and inflammation resulting in retinal ganglion cell (RGC) death. Remote ischemic post-conditioning (RIC) has been shown to enhance endogenous protective mechanisms in diverse disease models including stroke, vascular cognitive impairment (VCI), retinal injury and optic nerve injury. However, the protective mechanisms underlying the improvement of retinal function and RGC survival after RIC treatment remain unclear. Here, we hypothesized that RIC therapy may be protective following TON by preventing RGC death, oxidative insult and inflammation in the mouse retina. To carry out the study, mice were divided in three different groups (Control, TON and TON + RIC). We harvested retinal tissue 5 days after TON induction for western blotting and histochemical analysis. We observed increased TON-induced retinal cell death compared with controls by cleaved caspase-3 immunohistochemistry. Furthermore, the TON cohort demonstrated increased TUNEL positive cells which were significantly attenuated by RIC. Immunofluorescence data showed that oxidative stress markers dihydroethidium (DHE), NOX-2 and nitrotyrosine expression were elevated in the TON group relative to controls and RIC therapy significantly reduced the expression level of these markers. Next, we found that the proinflammatory cytokine TNF-α was increased and anti-inflammatory IL-10 was decreased in plasma of TON animals, and RIC therapy reversed this expression level. Interestingly, western blotting of retinal tissue showed that RGC marker Brn3a and tight junction proteins (ZO-1 and Occludin), and AMPKα1 expression were downregulated in the TON group compared to controls. However, RIC significantly increased the expression levels of these proteins. Together these data suggest that RIC therapy activates endogenous protective mechanisms which may attenuate TON-induced oxidative stress and inflammation, and improves BRB integrity.

Essential but partially redundant roles for POU4F1/Brn-3a and POU4F2/Brn-3b transcription factors in the developing heart.

Congenital heart defects contribute to embryonic or neonatal lethality but due to the complexity of cardiac development, the molecular changes associated with such defects are not fully understood. Here, we report that transcription factors (TFs) Brn-3a (POU4F1) and Brn-3b (POU4F2) are important for normal cardiac development. Brn-3a directly represses Brn-3b promoter in cardiomyocytes and consequently Brn-3a knockout (KO) mutant hearts express increased Brn-3b mRNA during mid-gestation, which is linked to hyperplastic growth associated with elevated cyclin D1, a known Brn-3b target gene. However, during late gestation, Brn-3b can cooperate with p53 to enhance transcription of pro-apoptotic genes e.g. Bax, thereby increasing apoptosis and contribute to morphological defects such as non-compaction, ventricular wall/septal thinning and increased crypts/fissures, which may cause lethality of Brn-3a KO mutants soon after birth. Despite this, early embryonic lethality in e9.5 double KO (Brn-3a-/- : Brn-3b-/-) mutants indicate essential functions with partial redundancy during early embryogenesis. High conservation between mammals and zebrafish (ZF) Brn-3b (87%) or Brn-3a (76%) facilitated use of ZF embryos to study potential roles in developing heart. Double morphant embryos targeted with morpholino oligonucleotides to both TFs develop significant cardiac defects (looping abnormalities and valve defects) suggesting essential roles for Brn-3a and Brn-3b in developing hearts.

Inherited Photoreceptor Degeneration Causes the Death of Melanopsin-Positive Retinal Ganglion Cells and Increases Their Coexpression of Brn3a.

PURPOSE: To study the population of intrinsically photosensitive retinal ganglion cells (melanopsin-expressing RGCs, m+RGCs) in P23H-1 rats, a rat model of inherited photoreceptor degeneration. METHODS: At postnatal (P) times P30, P365, and P540, retinas from P23H dystrophic rats (line 1, rapid degeneration; and line 3, slow degeneration) and Sprague Dawley (SD) rats (control) were dissected as whole-mounts and immunodetected for melanopsin and/or Brn3a. The dendritic arborization of m+RGCs and the numbers of Brn3a+RGCs and m+RGCs were quantified and their retinal distribution and coexpression analyzed. RESULTS: In SD rats, aging did not affect the population of Brn3a+RGCs or m+RGCs or the percentage that showed coexpression (0.27%). Young P23H-1 rats had a significantly lower number of Brn3a+RGCs and showed a further decline with age. The population of m+RGCs in young P23H-1 rats was similar to that found in SD rats and decreased by 22.6% and 28.2% at P365 and P540, respectively, similarly to the decrease of the Brn3a+RGCs. At these ages the m+RGCs showed a decrease of their dendritic arborization parameters, which was similar in both the P23H-1 and P23H-3 lines. The percentage of coexpression of Brn3a was, however, already significantly higher at P30 (3.31%) and increased significantly with age (10.65% at P540). CONCLUSIONS: Inherited photoreceptor degeneration was followed by secondary loss of Brn3a+RGCs and m+RGCs. Surviving m+RGCs showed decreased dendritic arborization parameters and increased coexpression of Brn3a and melanopsin, phenotypic and molecular changes that may represent an effort to resist degeneration and/or preferential survival of m+RGCs capable of synthesizing Brn3a.

The novel Rho kinase (ROCK) inhibitor K-115: a new candidate drug for neuroprotective treatment in glaucoma.

PURPOSE: To investigate the effect of K-115, a novel Rho kinase (ROCK) inhibitor, on retinal ganglion cell (RGC) survival in an optic nerve crush (NC) model. Additionally, to determine the details of the mechanism of K-115's neuroprotective effect in vivo and in vitro. METHODS: ROCK inhibitors, including K-115 and fasudil (1 mg/kg/d), or vehicle were administered orally to C57BL/6 mice. Retinal ganglion cell death was then induced with NC. Retinal ganglion cell survival was evaluated by counting surviving retrogradely labeled cells and measuring RGC marker expression with quantitative real-time polymerase chain reaction (qRT-PCR). Total oxidized lipid levels were assessed with a thiobarbituric acid-reactive substances (TBARS) assay. Reactive oxygen species (ROS) levels were assessed by co-labeling with CellROX and Fluorogold. Expression of the NADPH oxidase (Nox) family of genes was evaluated with qRT-PCR. RESULTS: The survival of RGCs after NC was increased 34 ± 3% with K-115, a significantly protective effect. Moreover, a similar effect was revealed by the qRT-PCR analysis of Thy-1.2 and Brn3a, RGC markers. Levels of oxidized lipids and ROS also increased with time after NC. NC-induced oxidative stress, including oxidation of lipids and production of ROS, was significantly attenuated by K-115. Furthermore, expression of the Nox gene family, especially Nox1, which is involved in the NC-induced ROS production pathway, was dramatically reduced by K-115. CONCLUSIONS: The results indicated that oral K-115 administration delayed RGC death. Although K-115 may be mediated through Nox1 downregulation, we found that it did not suppress ROS production directly. Our findings show that K-115 has a potential use in neuroprotective treatment for glaucoma and other neurodegenerative diseases.

Differential responses of neuronal and spermatogenic cells to the doppel cytotoxicity.

Although structurally and biochemically similar to the cellular prion (PrP(C)), doppel (Dpl) is unique in its biological functions. There are no reports about any neurodegenerative diseases induced by Dpl. However the artificial expression of Dpl in the PrP-deficient mouse brain causes ataxia with Purkinje cell death. Abundant Dpl proteins have been found in testis and depletion of the Dpl gene (Prnd) causes male infertility. Therefore, we hypothesize different regulations of Prnd in the nerve and male productive systems. In this study, by electrophoretic mobility shift assays we have determined that two different sets of transcription factors are involved in regulation of the Prnd promoter in mouse neuronal N2a and GC-1 spermatogenic (spg) cells, i.e., upstream stimulatory factors (USF) in both cells, Brn-3 and Sp1 in GC-1 spg cells, and Sp3 in N2a cells, leading to the expression of Dpl in GC-1 spg but not in N2a cells. We have further defined that, in N2a cells, Dpl induces oxidative stress and apoptosis, which stimulate ataxia-telangiectasia mutated (ATM)-modulating bindings of transcription factors, p53 and p21, to Prnp promoter, resulting the PrP(C) elevation for counteraction of the Dpl cytotoxicity; in contrast, in GC-1 spg cells, phosphorylation of p21 and N-terminal truncated PrP may play roles in the control of Dpl-induced apoptosis, which may benefit the physiological function of Dpl in the male reproduction system.

Decrease in retinal neuronal cells in streptozotocin-induced diabetic mice.

PURPOSE: Little is known about retinal neuronal loss in the retinas of diabetic mice. The purpose of this study was the quantitative assessment of retinal neural cell number in diabetic mice. METHODS: Five-week-old C57BL/6 mice were used as a diabetic model with streptozotocin. Mice were studied over the course of 6 and 12 weeks after the onset of diabetes. Intraocular pressure (IOP) was measured with a noninvasive TonoLab tonometer. The retinal ganglion cells (RGCs) were counted at two different time points after the induction of diabetes and examined using the immunofluorescence technique and quantitative analysis. RESULTS: The diabetic mice had significantly elevated IOP levels at 6 and 12 weeks after the onset of diabetes compared with the age-matched control mice (p<0.01 and p<0.001, respectively). The temporal course of Brn3a+ RGC and Neuronal Nuclei+RGC (NeuN+ RGC) loss induced by intraperitoneal injection of streptozotocin followed a similar trend. At 6 and 12 weeks after the onset of diabetes, the number of Brn3a+ RGCs (p<0.05 at 6 weeks; p<0.001 at 12 weeks) and NeuN+ RGCs (p<0.05 at 6 weeks; p<0.001 at 12 weeks) was significantly lower in diabetic mice than age-matched control mice. In the retinal flatmounts, the number of Brn3a+ RGCs (p<0.05 at 6 weeks, p<0.01 at 12 weeks) was also significantly lower in diabetic mice than control mice. The IOP in diabetic mice was negatively related with RGCs in cross sections. The cut-off value of IOP was 14.2 mmHg for diabetes. CONCLUSIONS: This is a specific quantitative study of neural cell loss in the retina during diabetes. These data suggest that retinal neural cell reduction occurs in diabetic mice. It indicates that RGC loss may be an important component of diabetic retinopathy.

Differential expression of Brn3 transcription factors in intrinsically photosensitive retinal ganglion cells in mouse.

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.

Missing optomotor head-turning reflex in the DBA/2J mouse.

PURPOSE: The optomotor reflex of DBA/2J (D2), DBA/2J-Gpnmb+ (D2-Gpnmb+), and C57BL/6J (B6) mouse strains was assayed, and the retinal ganglion cell (RGC) firing patterns, direction selectivity, vestibulomotor function and central vision was compared between the D2 and B6 mouse lines. METHODS: Intraocular pressure (IOP) measurements, real-time PCR, and immunohistochemical analysis were used to assess the time course of glaucomatous changes in D2 retinas. Behavioral analyses of optomotor head-turning reflex, visible platform Morris water maze and Rotarod measurements were conducted to test vision and vestibulomotor function. Electroretinogram (ERG) measurements were used to assay outer retinal function. The multielectrode array (MEA) technique was used to characterize RGC spiking and direction selectivity in D2 and B6 retinas. RESULTS: Progressive increase in IOP and loss of Brn3a signals in D2 animals were consistent with glaucoma progression starting after 6 months of age. D2 mice showed no response to visual stimulation that evoked robust optomotor responses in B6 mice at any age after eye opening. Spatial frequency threshold was also not measurable in the D2-Gpnmb+ strain control. ERG a- and b-waves, central vision, vestibulomotor function, the spiking properties of ON, OFF, ON-OFF, and direction-selective RGCs were normal in young D2 mice. CONCLUSIONS: The D2 strain is characterized by a lack of optomotor reflex before IOP elevation and RGC degeneration are observed. This behavioral deficit is D2 strain-specific, but is independent of retinal function and glaucoma. Caution is advised when using the optomotor reflex to follow glaucoma progression in D2 mice.

Organization of the human embryonic ventral mesencephalon.

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.

Brn-3a suppresses pseudorabies virus-induced cell death in sensory neurons.

Sensory neurons of the trigeminal ganglion (TG) are of crucial importance in the pathogenesis of many alphaherpesviruses, constituting major target cells for latency and reactivation events. We showed earlier that a subpopulation of porcine TG neurons, in contrast to other porcine cell types, is highly resistant to cell death induced by infection with the porcine alphaherpesvirus pseudorabies virus (PRV). Here, we report that expression of Brn-3a, a neuron-specific transcription factor implicated in cell survival of sensory neurons, correlates with the increased resistance of TG neurons towards PRV-induced cell death. In addition, overexpression of Brn-3a in the sensory neuronal cell line ND7 markedly increased resistance of these cells to PRV-induced cell death. Hence, Brn-3a may play a hitherto uncharacterized role in protection of sensory neurons from alphaherpesvirus-induced cell death, which may have implications for different aspects of the alphaherpesvirus life cycle, including latency/reactivation events.

Effects of Brn-3a protein and RNA expression in rat brain following low-level lead exposure during development on spatial learning and memory.

The developing nervous system is preferentially vulnerable to lead exposure with alterations in neuronal and glial cells of the brain. Chronic exposure to lead (Pb2+) causes deficits of learning and memory in children and spatial learning deficits in developing rats. Brn-3a is a member of the Pit-Oct-Unc (POU) family of transcription factors that is expressed predominantly in neuronal cells. It exists in two forms, with the long form containing 84 amino acids at the N-terminus that are lacking in the short form. The N-terminal domain unique to the long form induces expression of the Bcl-2 gene and protects neuronal cells against apoptosis whereas the C-terminal POU domain common to both forms is sufficient for activating a number of other neuronally expressed genes and stimulating neuronal process outgrowth. We examined Brn-3a protein and RNA expression in rat brain following low-level lead exposure during development and subsequent effects on spatial learning and memory. Two groups of rats were investigated: a control group and a lead-exposed group (0.2% lead acetate in the drinking water of the dam from gestational day 15 to postnatal day 21). Levels of Brn-3a were measured in rat cortex, hippocampus and cerebellum by immunohistochemistry and in situ hybridization, both protein and mRNA levels were reduced in lead-exposed group (p < 0.05). In Morris water maze, we found spatial learning deficits in rats of lead-exposed group (p < 0.05). These data suggest that the alteration of Brn-3a may play a key role in the mechanisms underlying lead neurotoxicity.

Brn3a-expressing retinal ganglion cells project specifically to thalamocortical and collicular visual pathways.

Retinal ganglion cells (RGCs) innervate several specific CNS targets serving cortical and subcortical visual pathways and the entrainment of circadian rhythms. Recent studies have shown that retinal ganglion cells express specific combinations of POU- and LIM-domain transcription factors, but how these factors relate to the subsequent development of the retinofugal pathways and the functional identity of RGCs is mostly unknown. Here, we use targeted expression of an genetic axonal tracer, tau/beta-galactosidase, to examine target innervation by retinal ganglion cells expressing the POU-domain factor Brn3a. Brn3a is expressed in RGCs innervating the principal retinothalamic/retinocollicular pathway mediating cortical vision but is not expressed in RGCs of the accessory optic, pretectal, and hypothalamic pathways serving subcortical visuomotor and circadian functions. In the thalamus, Brn3a ganglion cell fibers are primarily restricted to the outer shell of the dorsal lateral geniculate, providing new evidence for the regionalization of this nucleus in rodents. Brn3a RGC axons have a relative preference for the contralateral hemisphere, but known mediators of the laterality of RGC axons are not repatterned in the absence of Brn3a. Brn3a is coexpressed extensively with the closely related factor Brn3b in the embryonic retina, and the effects of the loss of Brn3a in retinal development are not severe, suggesting partial redundancy of function in this gene class.