Vascular dysfunction caused by loss of Brn-3b/POU4F2 transcription factor in aortic vascular smooth muscle cells is linked to deregulation of calcium signalling pathways.

Phenotypic and functional changes in vascular smooth muscle cells (VSMCs) contribute significantly to cardiovascular diseases (CVD) but factors driving early adverse vascular changes are poorly understood. We report on novel and important roles for the Brn-3b/POU4F2 (Brn-3b) transcription factor (TF) in controlling VSMC integrity and function. Brn-3b protein is expressed in mouse aorta with localisation to VSMCs. Male Brn-3b knock-out (KO) aortas displayed extensive remodelling with increased extracellular matrix (ECM) deposition, elastin fibre disruption and small but consistent narrowing/coarctation in the descending aortas. RNA sequencing analysis showed that these effects were linked to deregulation of genes required for calcium (Ca2+) signalling, vascular contractility, sarco-endoplasmic reticulum (S/ER) stress responses and immune function in Brn-3b KO aortas and validation studies confirmed changes in Ca2+ signalling genes linked to increased intracellular Ca2+ and S/ER Ca2+ depletion [e.g. increased, Cacna1d Ca2+ channels; ryanodine receptor 2, (RyR2) and phospholamban (PLN) but reduced ATP2a1, encoding SERCA1 pump] and chaperone proteins, Hspb1, HspA8, DnaJa1 linked to increased S/ER stress, which also contributes to contractile dysfunction. Accordingly, vascular rings from Brn-3b KO aortas displayed attenuated contractility in response to KCl or phenylephrine (PE) while Brn-3b KO-derived VSMC displayed abnormal Ca2+ signalling following ATP stimulation. This data suggests that Brn-3b target genes are necessary to maintain vascular integrity /contractile function and deregulation upon loss of Brn-3b will contribute to contractile dysfunction linked to CVD.

Improved urine DNA methylation panel for early bladder cancer detection.

BACKGROUND: Bladder cancer is one of the most common malignancies but the corresponding diagnostic methods are either invasive or limited in specificity and/or sensitivity. This study aimed to develop a urine-based methylation panel for bladder cancer detection by improving published panels and validate performance of the new panel with clinical samples. METHODS: Related researches were reviewed and 19 potential panels were selected. RRBS was performed on a cohort with 45 samples to reassess these panels and a new panel inherited best markers was developed. The new panel was applied with qMSP platform to 33 samples from the RRBS cohort and the results were compared to those of RRBS. Lastly, another larger cohort with 207 samples was used to validate new panel performance with qMSP. RESULTS: Three biomarkers (PCDH17, POU4F2 and PENK) were selected to construct a new panel P3. P3 panel achieved 100% specificity and 71% sensitivity with RRBS in corresponding cohort and then showed a better performance of 100% specificity and 84% sensitivity with qMSP platforms in a balanced cohort. When validated with 207-sample cohort, P3 with qMSP showed a performance of 97% specificity and 87% sensitivity which was modestly improved compared to the panels it derided from. CONCLUSIONS: Overall, the P3 panel achieved relatively high sensitivity and accuracy in bladder cancer detection.

Inhibition of the cysteinyl leukotriene pathways increases survival of RGCs and reduces microglial activation in ocular hypertension.

Glaucoma is the second leading cause of blindness worldwide. This multifactorial, neurodegenerative group of diseases is characterized by the progressive loss of retinal ganglion cells (RGCs) and their axons, leading to irreversible visual impairment and blindness. There is a huge unmet and urging need for the development of new and translatable strategies and treatment options to prevent this progressive loss of RGC. Accumulating evidence points towards a critical role of neuroinflammation, in particular microglial cells, in the pathogenesis of glaucoma. Leukotrienes are mediators of neuroinflammation and are involved in many neurodegenerative diseases. Therefore, we tested the leukotriene receptors CysLT1R/GPR17-selective antagonist Montelukast (MTK) for its efficacy to modulate the reactive state of microglia in order to ameliorate RGCs loss in experimental glaucoma. Ocular hypertension (OHT) was induced unilaterally by injection of 8 µm magnetic microbead (MB) into the anterior chamber of female Brown Norway rats. The contralateral, untreated eye served as control. Successful induction of OHT was verified by daily IOP measurement using a TonoLab rebound tonometer. Simultaneously to OHT induction, one group received daily MTK treatment and the control group vehicle solution by oral gavage. Animals were sacrificed 13-15 days after MB injection. Retina and optic nerves (ON) of OHT and contralateral eyes were analyzed by immunofluorescence with specific markers for RGCs (Brn3a), microglial cells/macrophages (Iba1 and CD68), and cysteinyl leukotriene pathway receptors (CysLT1R and GPR17). Protein labeling was documented by confocal microscopy and analyzed with ImageJ plugins. Further, mRNA expression of genes of the inflammatory and leukotriene pathway was analyzed in retinal tissue. MTK treatment resulted in a short-term IOP reduction at day 2, which dissipated by day 5 of OHT induction in MTK treated animals. Furthermore, MTK treatment resulted in a decreased activation of Iba1+ microglial cells in the retina and ON, and in a significantly increased RGC survival in OHT eyes. Within the retina, GPR17 and CysLT1R expression was demonstrated in single RCGs and in microglial cells respectively. Further, increased mRNA expression of pro-inflammatory genes was detected in OHT induced retinas. In the ON, OHT induction increased the number of GPR17+ cells, showing a trend of reduction following MTK treatment. This study shows for the first time a significantly increased RGC survival in an acute OHT model following treatment with the leukotriene receptor antagonist MTK. These results strongly suggest a neuroprotective effect of MTK and a potential new therapeutic strategy for glaucoma treatment.

Transcription factor POU4F2 promotes colorectal cancer cell migration and invasion through hedgehog-mediated epithelial-mesenchymal transition.

As a POU homeodomain transcription factor, POU4F2 has been implicated in regulating tumorigenic processes in various cancers. However, the role of POU4F2 in colorectal cancer (CRC) remains unclear. Here, we revealed that POU4F2 functions as a tumor promotor in CRC. Bioinformatics analysis in specimens from CRC patients and expression analysis in CRC cell lines showed that POU4F2 was upregulated at the mRNA and protein levels in CRC. Depletion of POU4F2 suppressed the metastatic phenotypes of CRC cells, including cell migration, invasion, and the expression of epithelial-mesenchymal transition (EMT) markers. Moreover, depletion of POU4F2 decreased the number of lung metastatic nodes in nude mice. Mechanistically, POU4F2 positively regulated the Hedgehog signaling pathway, as inferred from the downregulation of the expression of sonic Hedgehog homolog, patched 1, Smoothened, and GLI family zinc finger 1 in vitro and vivo following silencing of POU4F2. Furthermore, the SMO agonist SAG reversed the effects of POU4F2 knockdown in CRC. Functionally, POU4F2 contributed to the Hedgehog signaling-regulated activation of the EMT process and promotion of CRC cell migration and invasion. Collectively, these findings elucidated the role of POU4F2 as a tumor promotor in CRC through the regulation of Hedgehog signaling-mediated EMT and suggested that POU4F2 suppression might be a promising therapeutic target in inhibiting CRC metastasis.

Linking metabolic dysfunction with cardiovascular diseases: Brn-3b/POU4F2 transcription factor in cardiometabolic tissues in health and disease.

Metabolic and cardiovascular diseases are highly prevalent and chronic conditions that are closely linked by complex molecular and pathological changes. Such adverse effects often arise from changes in the expression of genes that control essential cellular functions, but the factors that drive such effects are not fully understood. Since tissue-specific transcription factors control the expression of multiple genes, which affect cell fate under different conditions, then identifying such regulators can provide valuable insight into the molecular basis of such diseases. This review explores emerging evidence that supports novel and important roles for the POU4F2/Brn-3b transcription factor (TF) in controlling cellular genes that regulate cardiometabolic function. Brn-3b is expressed in insulin-responsive metabolic tissues (e.g. skeletal muscle and adipose tissue) and is important for normal function because constitutive Brn-3b-knockout (KO) mice develop profound metabolic dysfunction (hyperglycaemia; insulin resistance). Brn-3b is highly expressed in the developing hearts, with lower levels in adult hearts. However, Brn-3b is re-expressed in adult cardiomyocytes following haemodynamic stress or injury and is necessary for adaptive cardiac responses, particularly in male hearts, because male Brn-3b KO mice develop adverse remodelling and reduced cardiac function. As a TF, Brn-3b regulates the expression of multiple target genes, including GLUT4, GSK3ß, sonic hedgehog (SHH), cyclin D1 and CDK4, which have known functions in controlling metabolic processes but also participate in cardiac responses to stress or injury. Therefore, loss of Brn-3b and the resultant alterations in the expression of such genes could potentially provide the link between metabolic dysfunctions with adverse cardiovascular responses, which is seen in Brn-3b KO mutants. Since the loss of Brn-3b is associated with obesity, type II diabetes (T2DM) and altered cardiac responses to stress, this regulator may provide a new and important link for understanding how pathological changes arise in such endemic diseases.

Single cell transcriptomics reveals lineage trajectory of retinal ganglion cells in wild-type and Atoh7-null retinas.

Atoh7 has been believed to be essential for establishing the retinal ganglion cell (RGC) lineage, and Pou4f2 and Isl1 are known to regulate RGC specification and differentiation. Here we report our further study of the roles of these transcription factors. Using bulk RNA-seq, we identify genes regulated by the three transcription factors, which expand our understanding of the scope of downstream events. Using scRNA-seq on wild-type and mutant retinal cells, we reveal a transitional cell state of retinal progenitor cells (RPCs) co-marked by Atoh7 and other genes for different lineages and shared by all early retinal lineages. We further discover the unexpected emergence of the RGC lineage in the absence of Atoh7. We conclude that competence of RPCs for different retinal fates is defined by lineage-specific genes co-expressed in the transitional state and that Atoh7 defines the RGC competence and collaborates with other factors to shepherd transitional RPCs to the RGC lineage.

Diversity of intrinsically photosensitive retinal ganglion cells: circuits and functions.

The melanopsin-expressing, intrinsically photosensitive retinal ganglion cells (ipRGCs) are a relatively recently discovered class of atypical ganglion cell photoreceptor. These ipRGCs are a morphologically and physiologically heterogeneous population that project widely throughout the brain and mediate a wide array of visual functions ranging from photoentrainment of our circadian rhythms, to driving the pupillary light reflex to improve visual function, to modulating our mood, alertness, learning, sleep/wakefulness, regulation of body temperature, and even our visual perception. The presence of melanopsin as a unique molecular signature of ipRGCs has allowed for the development of a vast array of molecular and genetic tools to study ipRGC circuits. Given the emerging complexity of this system, this review will provide an overview of the genetic tools and methods used to study ipRGCs, how these tools have been used to dissect their role in a variety of visual circuits and behaviors in mice, and identify important directions for future study.

Quick Commitment and Efficient Reprogramming Route of Direct Induction of Retinal Ganglion Cell-like Neurons.

Direct reprogramming has been widely explored to generate various types of neurons for neurobiological research and translational medicine applications, but there is still no efficient reprogramming method to generate retinal ganglion cell (RGC)-like neurons, which are the sole projection neurons in the retina. Here, we show that three transcription factors, Ascl1, Brn3b, and Isl1, efficiently convert fibroblasts into RGC-like neurons (iRGCs). Furthermore, we show that the competence of cells to enter iRGC reprogramming route is determined by the cell-cycle status at a very early stage of the process. The iRGC reprogramming route involves intermediate states that are characterized by a transient inflammatory-like response followed by active epigenomic and transcriptional modifications. Our study provides an efficient method to generate iRGCs, which would be a valuable cell source for potential glaucoma cell replacement therapy and drug screening studies, and reveals the key cellular events that govern successful neuronal fate reprogramming.

Neuroprotective effects of Acer palmatum thumb. leaf extract (KIOM-2015E) against ischemia/reperfusion-induced injury in the rat retina.

Purpose: The present study aimed to determine whether the administration of Acer palmatum thumb. leaf extract (KIOM-2015E) protects against the degeneration of rat retinal ganglion cells after ischemia/reperfusion (I/R) induced by midbrain cerebral artery occlusion (MCAO). Methods: Sprague-Dawley rats were subjected to 90 min of MCAO, which produces transient ischemia in both the retina and brain due to the use of an intraluminal filament that blocks the ophthalmic and middle cerebral arteries. This was followed by reperfusion under anesthesia with isoflurane. The day after surgery, the eyes were treated three times (eye drop) or one time (oral administration) daily with KIOM-2015E for five days. Retinal histology was assessed in flat mounts and vertical sections to determine the effect of KIOM-2015E on I/R injury. Results: A significant loss of brain-specific homeobox/POU domain protein 3A (Brn3a) and neuron-specific class III beta-tubulin (Tuj-1) fluorescence and a marked increase in glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS) expression were observed after five days in the PBS-treated MCAO group compared to the sham-operated control group. However, KIOM-2015E treatment reduced (1) MCAO-induced upregulation of GFAP and GS, (2) retinal ganglion cell loss, (3) nerve fiber degeneration, and (4) the number of TUNEL-positive cells. KIOM-2015E application also increased staining for parvalbumin (a marker of horizontal cell associated calcium-binding protein and amacrine cells) and recoverin (a marker of photoreceptor expression) in rats subjected to MCAO-induced retinal damage. Conclusions: Our findings indicated that KIOM-2015E treatment exerted protective effects against retinal damage following MCAO injury and that this extract may aid in the development of novel therapeutic strategies for retinal diseases, such as glaucoma and age-related macular disease.

Retinal ganglion cell defects cause decision shifts in visually evoked defense responses.

A variety of visual cues can trigger defensive reactions in mice and other species. In mice, looming stimuli that mimic an approaching aerial predator elicit flight or freezing reactions, while sweeping stimuli that mimic an aerial predator flying parallel to the ground typically elicit freezing. The retinal ganglion cell (RGC) types involved in these circuits are largely unknown. We previously discovered that loss of RGC subpopulations in Brn3b knockout mice results in distinct visual response deficits. Here, we report that retinal or global loss of Brn3b selectively ablates the fleeing response to looming stimuli while leaving the freeze response intact. In contrast, freezing responses to sweeping stimuli are significantly affected. Genetic manipulations removing three RGC subpopulations (Brn3a+ betta RGCs, Opn4+Brn3b+, and Brn3c+Brn3b+ RGCs) result in milder phenocopies of Brn3b knockout response deficits. These findings show that flight and freezing responses to distinct visual cues are mediated by circuits that can already be separated at the level of the retina, potentially by enlisting dedicated RGC types.NEW & NOTEWORTHY Flight and freezing response choices evoked by visual stimuli are controlled by brain stem and thalamic circuits. Genetically modified mice with loss of specific retinal ganglion cell (RGC) subpopulations have altered flight versus freezing choices in response to some but not other visual stimuli. This finding suggests that "threatening" visual stimuli may be computed already at the level of the retina and communicated via dedicated pathways (RGCs) to the brain.

Two new genetically modified mouse alleles labeling distinct phases of retinal ganglion cell development by fluorescent proteins.

BACKGROUND: During development, all retinal cell types arise from retinal progenitor cells (RPCs) in a step-wise fashion. Atoh7 and Pou4f2 mark, and function in, two phases of retinal ganglion cell (RGC) genesis; Atoh7 functions in a subpopulation of RPCs to render them competent for the RGC fate, whereas Pou4f2 participates in RGC fate specification and RGC differentiation. Despite extensive research on their roles, the properties of the two phases represented by these two factors have not been well studied, likely due to the retinal cellular heterogeneity. RESULTS: In this report, we describe two novel knock-in mouse alleles, Atoh7zsGreenCreERT2 and Pou4f2FlagtdTomato , which labeled retinal cells in the two phases of RGC development by fluorescent proteins. Also, the Atoh7zsGreenCreERT2 allele allowed for indirect labeling of RGCs and other cell types upon tamoxifen induction in a dose-dependent manner. Further, these alleles could be used to purify retinal cells in the different phases by fluorescence assisted cell sorting (FACS). Single cell RNA-seq analysis of purified cells from Atoh7zsGreenCreERT2 retinas further validated that this allele labeled both transitional/competent RPCs and their progenies including RGCs. CONCLUSIONS: Thus, these two alleles are very useful tools for studying the molecular and genetic mechanisms underlying RGC formation.

No association between POU4F1, POU4F2, ISL1 polymorphisms and normal-tension glaucoma.

BACKGROUND: Normal-tension glaucoma (NTG) that occurs despite normal intraocular pressure has genetic predisposition. Since retinal ganglion cells (RGCs) are a key node in pathogenesis of glaucoma, neurodegeneration of RGCs is thought to be the main cause increasing the risk of NTG development. Here, we aimed to investigate the association of polymorphisms in RGC development genes with NTG development. MATERIALS AND METHODS: We performed a case-control association study of 435 patients with NTG and 419 normal controls. We genotyped four single nucleotide polymorphisms (SNPs) in genes responsible for RGC development, namely POU4F2 (rs13152799 and rs1504360), POU4F1 (rs9601092), and ISL1 (rs2288468), by either real-time PCR or PCR-RFLP, and evaluated its association with the risk of NTG development. RESULTS: No significant association was observed between the candidate SNPs and NTG development. CONCLUSIONS: To the best of our knowledge, this is the first report exploring the association between genes regulating RGC development and NTG susceptibility. Our data could provide a reference for further researches that focus on finding additional potential SNPs of POU4F2, POU4F1, ISL1 or other RGC development genes for NTG.

nGnG Amacrine Cells and Brn3b-negative M1 ipRGCs are Specifically Labeled in the ChAT-ChR2-EYFP Mouse.

Purpose: Experimental access to specific cell subtypes is essential for deciphering the complexity of retinal networks. Here, we characterized the selective labeling, caused by ectopic transgene expression, of two atypical retinal neurons in the ChAT-Channelrhodopsin-2 (ChR2)-EYFP mouse. Methods: Retinal sections and flat-mounts were prepared for double-staining immunohistochemistry with antibodies against EYFP and various neuronal markers. Sagittal/coronal brain slices were made to visualize EYFP signals in central nuclei. Whole-cell recordings were conducted to test the functionality of ChR2. Results: Two populations of EYFP-positive retinal cells were observed. The inner nuclear layer (INL)-located one (type I cell) distributed regularly throughout the entire retina, whereas the ganglion cell layer (GCL)-residing one (type II cell) was restricted ventrally. None of them was cholinergic, as evidenced by the complete absence of ChAT immunoreactivity. Type I cells were immunolabeled by the amacrine marker syntaxin. However, the vast majority of them were neither positive to GABA/GAD65, nor to GlyT1/glycine, suggesting that they were non-GABAergic non-glycinergic amacrine cells (nGnG ACs), which was confirmed by double-labeling with the nGnG AC marker PPP1R17. Type II cells were immunopositive to melanopsin, but not to Brn3a or Brn3b. They possessed dendrites stratifying in the outermost inner plexiform layer (IPL) and axons projecting to the suprachiasmatic nucleus (SCN) rather than the olivary pretectal nucleus (OPN), suggesting that they belonged to a Brn3b-negative subset of M1-type intrinsically photosensitive retinal ganglion cells (ipRGCs). Glutamatergic transmission-independent photocurrents were elicited in EYFP-positive cells, indicating the functional expression of ChR2. Conclusions: The ChAT-ChR2-EYFP retina exhibits ectopic, but functional, transgene expression in nGnG ACs and SCN-innervating M1 ipRGCs, thus providing an ideal tool to achieve efficient labeling and optogenetic manipulation of these cells.

The emergence of transcriptional identity in somatosensory neurons.

More than twelve morphologically and physiologically distinct subtypes of primary somatosensory neuron report salient features of our internal and external environments1-4. It is unclear how specialized gene expression programs emerge during development to endow these subtypes with their unique properties. To assess the developmental progression of transcriptional maturation of each subtype of principal somatosensory neuron, we generated a transcriptomic atlas of cells traversing the primary somatosensory neuron lineage in mice. Here we show that somatosensory neurogenesis gives rise to neurons in a transcriptionally unspecialized state, characterized by co-expression of transcription factors that become restricted to select subtypes as development proceeds. Single-cell transcriptomic analyses of sensory neurons from mutant mice lacking transcription factors suggest that these broad-to-restricted transcription factors coordinate subtype-specific gene expression programs in subtypes in which their expression is maintained. We also show that neuronal targets are involved in this process; disruption of the prototypic target-derived neurotrophic factor NGF leads to aberrant subtype-restricted patterns of transcription factor expression. Our findings support a model in which cues that emanate from intermediate and final target fields promote neuronal diversification in part by transitioning cells from a transcriptionally unspecialized state to transcriptionally distinct subtypes by modulating the selection of subtype-restricted transcription factors.

HOXA9, PCDH17, POU4F2, and ONECUT2 as a Urinary Biomarker Combination for the Detection of Bladder Cancer in Chinese Patients with Hematuria.

BACKGROUND: DNA methylation biomarkers for bladder cancer (BCa) have not been evaluated extensively in the Chinese population. OBJECTIVE: To develop and validate a urinary biomarker combination of methylation assays in a group of Chinese patients with hematuria. DESIGN, SETTING, AND PARTICIPANTS: A total of 192 urine samples were collected and evaluated from patients with microscopic or gross hematuria, including 97 BCa patients and 95 controls with benign diseases. A two-stage study was conducted: the first stage being assay construction and the second stage being assay validation. Eighty-one urine samples were analyzed for the hypermethylation of eight selected genes in stage 1 and then a four-gene panel was constructed. An additional 111 urine samples were analyzed using the four-gene panel (including HOXA9, PCDH17, POU4F2, and ONECUT2) for independent validation. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The positive predictive value (PPV) and negative predictive value (NPV) were calculated for the combination methylation assay. Uni- and multivariate binary logistic regression analyses (backward elimination, conditional) were performed to calculate the association between BCa and each predictor variable. RESULTS AND LIMITATIONS: The combination assay of HOXA9, PCDH17, POU4F2, and ONECUT2 was selected based on the results of multivariate logistic regression analysis in stage 1. Using a strategy of three-level risk stratification, the assay yielded a consistent PPV of 100%. With an estimated BCa prevalence of 10% in a general hematuria population, the assay would result in an overall NPV of 98%. This combined methylation biomarker would yield an overall area under the receiver operating characteristic curve of 0.871 (with a sensitivity of 90.5% and a specificity of 73.2%) if using the prediction model from multivariate regression analysis. In addition, over half of BCa cases would be predicted accurately and ∼60% of unnecessary cystoscopies could be spared. This study had several limitations. First, the sample size was relatively small. Second, it was performed in a case-control population rather than in a natural hematuria cohort. CONCLUSIONS: A combination methylation assay of HOXA9, PCDH17, POU4F2, and ONECUT2 resulted in high PPV and NPV in Chinese patients with hematuria. With accurate risk prediction, the urinary biomarker combination could spare a sizeable proportion of low-risk patients from extensive and invasive examination. PATIENT SUMMARY: In the present study, we looked at the predictive performance of a urinary biomarker combination of HOXA9, PCDH17, POU4F2, and ONECUT2. We found that this urinary biomarker combination may help discriminate bladder cancer from other benign diseases in patients with hematuria, resulting in a reduction of unnecessary invasive examination in patients at low risk.

Transplantation of Retinal Ganglion Cells Derived from Male Germline Stem Cell as a Potential Treatment to Glaucoma.

Glaucoma is characterized by retinal ganglion cell (RGC) degeneration and is the second leading cause of blindness worldwide. However, current treatments such as eye drop or surgery have limitations and do not target the loss of RGC. Regenerative therapy using embryonic stem cells (ESCs) holds a promising option, but ethical concern hinders clinical applications on human subjects. In this study, we employed spermatogonial stem cells (SSCs) as an alternative source of ESCs for cell-based regenerative therapy in mouse glaucoma model. We generated functional RGCs from SSCs with a two-step protocol without applying viral transfection or chemical induction. SSCs were first dedifferentiated to embryonic stem-like cells (SSC-ESCs) that resemble ESCs in morphology, gene expression signatures, and stem cell properties. The SSC-ESCs then differentiated toward retinal lineages. We showed SSC-ESC-derived retinal cells expressed RGC-specific marker Brn3b and functioned as bona fide RGCs. To allow in vivo RGC tracing, Brn3b-EGFP reporter SSC-ESCs were generated and the derived RGCs were subsequently transplanted into the retina of glaucoma mouse models by intravitreal injection. We demonstrated that the transplanted RGCs could survive in host retina for at least 10 days after transplantation. SSC-ESC-derived RGCs can thus potentially be a novel alternative to replace the damaged RGCs in glaucomatous retina.

De novo genesis of retinal ganglion cells by targeted expression of Klf4 in vivo.

Retinal ganglion cell (RGC) degeneration is a hallmark of glaucoma, the most prevalent cause of irreversible blindness. Thus, therapeutic strategies are needed to protect and replace these projection neurons. One innovative approach is to promote de novo genesis of RGCs via manipulation of endogenous cell sources. Here, we demonstrate that the pluripotency regulator gene Krüppel-like factor 4 (Klf4) is sufficient to change the potency of lineage-restricted retinal progenitor cells to generate RGCs in vivo Transcriptome analysis disclosed that the overexpression of Klf4 induces crucial regulators of RGC competence and specification, including Atoh7 and Eya2 In contrast, loss-of-function studies in mice and zebrafish demonstrated that Klf4 is not essential for generation or differentiation of RGCs during retinogenesis. Nevertheless, induced RGCs (iRGCs) generated upon Klf4 overexpression migrate to the proper layer and project axons aligned with endogenous fascicles that reach the optic nerve head. Notably, iRGCs survive for up to 30 days after in vivo generation. We identified Klf4 as a promising candidate for reprogramming retinal cells and regenerating RGCs in the retina.This article has an associated 'The people behind the papers' interview.

The POU4F2/Brn-3b transcription factor is required for the hypertrophic response to angiotensin II in the heart.

Adult hearts respond to increased workload such as prolonged stress or injury, by undergoing hypertrophic growth. During this process, the early adaptive responses are important for maintaining cardiac output whereas at later stages, pathological responses such as cardiomyocyte apoptosis and fibrosis cause adverse remodelling, that can progress to heart failure. Yet the factors that control transition from adaptive responses to pathological remodelling in the heart are not well understood. Here we describe the POU4F2/Brn-3b transcription factor (TF) as a novel regulator of adaptive hypertrophic responses in adult hearts since Brn-3b mRNA and protein are increased in angiotensin-II (AngII) treated mouse hearts with concomitant hypertrophic changes [increased heart weight:body weight (HW:BW) ratio]. These effects occur specifically in cardiomyocytes because Brn-3b expression is increased in AngII-treated primary cultures of neonatal rat ventricular myocytes (NRVM) or foetal heart-derived H9c2 cells, which undergo characteristic sarcomeric re-organisation seen in hypertrophic myocytes and express hypertrophic markers, ANP/ßMHC. The Brn-3b promoter is activated by known hypertrophic signalling pathways e.g. p42/p44 mitogen-activated protein kinase (MAPK/ERK1/2) or calcineurin (via NFAT). Brn-3b target genes, e.g. cyclin D1, GLUT4 and Bax, are increased at different stages following AngII treatment, supporting distinct roles in cardiac responses to stress. Furthermore, hearts from male Brn-3b KO mutant mice display contractile dysfunction at baseline but also attenuated hypertrophic responses to AngII treatment. Hearts from AngII-treated male Brn-3b KO mice develop further contractile dysfunction linked to extensive fibrosis/remodelling. Moreover, known Brn-3b target genes, e.g. GLUT4, are reduced in AngII-treated Brn-3b KO hearts, suggesting that Brn-3b and its target genes are important in driving adaptive hypertrophic responses in stressed heart.

A Novel Reporter Mouse Uncovers Endogenous Brn3b Expression.

Brn3b (Pou4f2) is a class-4 POU domain transcription factor known to play central roles in the development of different neuronal populations of the Central Nervous System, including retinal ganglion cells (RGCs), the neurons that connect the retina with the visual centers of the brain. Here, we have used CRISPR-based genetic engineering to generate a Brn3b-mCherry reporter mouse without altering the endogenous expression of Brn3b. In our mouse line, mCherry faithfully recapitulates normal Brn3b expression in the retina, the optic tracts, the midbrain tectum, and the trigeminal ganglia. The high sensitivity of mCherry also revealed novel expression of Brn3b in the neuroectodermal cells of the optic stalk during early stages of eye development. Importantly, the fluorescent intensity of Brn3b-mCherry in our reporter mice allows for noninvasive live imaging of RGCs using Scanning Laser Ophthalmoscopy (SLO), providing a novel tool for longitudinal monitoring of RGCs.

Simultaneous co-delivery of neuroprotective drugs from multi-loaded PLGA microspheres for the treatment of glaucoma.

Glaucoma is a multifactorial neurodegenerative disorder and one of the leading causes of irreversible blindness globally and for which intraocular pressure is the only modifiable risk factor. Although neuroprotective therapies have been suggested to have therapeutic potential, drug delivery for the treatment of ocular disorders such as glaucoma remains an unmet clinical need, further complicated by poor patient compliance with topically applied treatments. In the present study we describe the development of multi-loaded PLGA-microspheres (MSs) incorporating three recognised neuroprotective agents (dexamethasone (DX), melatonin (MEL) and coenzyme Q10 (CoQ10)) in a single formulation (DMQ-MSs) to create a novel sustained-release intraocular drug delivery system (IODDS) for the treatment of glaucoma. MSs were spherical, with a mean particle size of 29.04 ±â€¯1.89 µm rendering them suitable for intravitreal injection using conventional 25G-32G needles. >62% incorporation efficiency was achieved for the three drug cargo and MSs were able to co-deliver the encapsulated active compounds in a sustained manner over 30-days with low burst release. In vitro studies showed DMQ-MSs to be neuroprotective in a glutamate-induced cytotoxicity model (IC50 10.00 ±â€¯0.94 mM versus 6.89 ±â€¯0.82 mM in absence of DMQ-MSs) in R28 cell line. In vivo efficacy studies were performed using a well-established rodent model of chronic ocular hypertension (OHT), comparing single intravitreal injections of microspheres of DMQ-MSs to their equivalent individual single-drug loaded MSs mixture (MSsmix), empty MSs, no-treatment OHT only and naïve groups. Twenty one days after OHT induction, DMQ-MSs showed a significantly neuroprotective effect on RGCs compared to OHT only controls. No such protective effect was observed in empty MSs and single-drug MSs treated groups. This work suggests that multi-loaded PLGA MSs present a novel therapeutic approach in the management of retinal neurodegeneration conditions such as glaucoma.