Repeat Brn3a immunolabeling rescues faded staining and improves detection of retinal ganglion cells.

Immunofluorescence is used in numerous research areas including eye research to detect specific antigens in cells and tissues. One limitation is that fluorescent signal can fade, causing detection problems if data recording was not completed in a timely manner or if additional data acquisition is required. The ability to repeat immunostaining for the same antigen after initial fluorescence has faded may require time-consuming and potentially damaging steps to remove primary antibodies. Our studies assessed whether immunofluorescence could be reapplied to previously labeled retinal ganglion cells (RGCs). To examine whether immunostaining of Brn3a, a commonly used RGC marker, could be repeated in retinas with previously faded immunostaining, retinal whole mounts were labeled with anti-Brn3a primary antibodies and green fluorescent secondary antibodies, then allowed to fade over time. Faded retinas were restained with anti-Brn3a antibody followed by secondary antibody, or with secondary antibody alone. Results show restaining with anti-Brn3a primary antibody followed by Alexa-fluor green secondary antibody is effective for RGC detection. Repeat RGC labeling improved the clarity of staining compared with original staining prior to fading, with significant reduction in the percentage of blurry/out of focus fluorescent cells (6 vs 26%); whereas, repeat application of secondary antibody alone was not effective. Preflattening retinas under a coverslip prior to initial Brn3a staining also increased the clarity of staining, and facilitated significantly more accurate automated counting of RGCs. Findings suggest Brn3a antigen remains accessible for repeat immunofluorescence labeling after original staining fades. Staining retinas after flattening tissue may enhance the clarity of staining and accuracy of automated RGC counting. Repeat immunofluorescence staining, without the need to strip off prior bound antibodies, may be useful in other tissues as well and warrants future examination.

HDAC2 Is Involved in the Regulation of BRN3A in Melanocytes and Melanoma.

The neural crest transcription factor BRN3A is essential for the proliferation and survival of melanoma cells. It is frequently expressed in melanoma but not in normal melanocytes or benign nevi. The mechanisms underlying the aberrant expression of BRN3A are unknown. Here, we investigated the epigenetic regulation of BRN3A in melanocytes and melanoma cell lines treated with DNA methyltransferase (DNMT), histone acetyltransferase (HAT), and histone deacetylase (HDAC) inhibitors. DNMT and HAT inhibition did not significantly alter BRN3A expression levels, whereas panHDAC inhibition by trichostatin A led to increased expression. Treatment with the isoform-specific HDAC inhibitor mocetinostat, but not with PCI-34051, also increased BRN3A expression levels, suggesting that class I HDACs HDAC1, HDAC2, and HDAC3, and class IV HDAC11, were involved in the regulation of BRN3A expression. Transient silencing of HDACs 1, 2, 3, and 11 by siRNAs revealed that, specifically, HDAC2 inhibition was able to increase BRN3A expression. ChIP-Seq analysis uncovered that HDAC2 inhibition specifically increased H3K27ac levels at a distal enhancer region of the BRN3A gene. Altogether, our data suggest that HDAC2 is a key epigenetic regulator of BRN3A in melanocytes and melanoma cells. These results highlight the importance of epigenetic mechanisms in regulating melanoma oncogenes.

Chronic social defeat stress causes retinal vascular dysfunction.

PURPOSE: The roles of vascular dysfunction and chronic stress have been extensively discussed in the pathophysiology of glaucoma. Our aim was to test whether chronic stress causes retinal vascular dysfunction and therewith induces retinal ganglion cells (RGCs) loss. METHODS: Twelve mice underwent chronic social defeat (CSD) stress, while 12 mice received control treatment only. Intraocular pressure (IOP) was measured with a rebound tonometer. Blood plasma corticosterone concentration and adrenal gland weight were used to assess stress levels. Brn-3a staining in retinas and PPD staining in optic nerve cross sections were conducted to assess the survival of RGCs and axons respectively. The ET-1 and α-SMA levels were determined in retina. Retinal vascular autoregulation, functional response to various vasoactive agents and vascular mechanics were measured using video microscopy. RESULTS: No significant difference in IOP levels was observed during and after CSD between CSD mice and controls. CSD stress caused hypercortisolemia 2 days post-CSD. However, increased corticosterone levels went back to normal 8 months after CSD. CSD-exposed mice developed adrenal hyperplasia 3 days post-CSD, which was normalized by 8 months. RGC and axon survival were similar between CSD mice and controls. However, CSD stress caused irreversible, impaired autoregulation and vascular dysfunction of retinal arterioles in CSD mice. In addition, impaired maximal dilator capacity of retinal arterioles was observed 8 months post-CSD rather than 3 days post-CSD. Remarkably, ET-1 levels were increased 3 days post-CSD while α-SMA levels were decreased 8 months post-CSD. CONCLUSIONS: We found that CSD stress does not cause IOP elevation, nor loss of RGCs and their axons. However, it strikingly causes irreversible impaired autoregulation and endothelial function in murine retinal arterioles. In addition, CSD changed vascular mechanics on a long-term basis. Increased ET-1 levels and loss of pericytes in retina vessels may involve in this process.

An In Vitro Model of Diabetic Retinal Vascular Endothelial Dysfunction and Neuroretinal Degeneration.

BACKGROUND: Diabetic retinopathy (DR) is a leading cause of blindness in working-age populations. Proper in vitro DR models are crucial for exploring pathophysiology and identifying novel therapeutic targets. This study establishes a rational in vitro diabetic retinal neuronal-endothelial dysfunction model and a comprehensive downstream validation system. METHODS: Human retinal vascular endothelial cells (HRMECs) and retinal ganglion cells (RGCs) were treated with different glucose concentrations with mannitol as matched osmotic controls. Cell proliferation and viability were evaluated by the Cell Counting Kit-8. Cell migration was measured using a transwell migration assay. Cell sprouting was assessed by a tube formation assay. The VEGF expression was assessed by ELISA. RGCs were labeled by neurons and RGC markers TUJ1 and BRN3A for quantitative and morphological analysis. Apoptosis was detected using PI/Hoechst staining and TUNEL assay and quantified by ImageJ. RESULTS: Cell proliferation and migration in HRMECs were significantly higher in the 25 mM glucose-treated group (p < 0.001) but lower in the 50 mM and 100 mM groups (p < 0.001). The permeability and the apoptotic index in HRMECs were statistically higher in the 25 mM, 50 mM, and 100 mM groups (p < 0.05). The tube formation assay found that all the parameters were significantly higher in the 25 mM and 50 mM groups (p < 0.001) concomitant with the elevated VEGFA expression in HRMECs (p = 0.016). Cell viability was significantly lower in the 50 mM, 100 mM, and 150 mM groups in RGCs (p 50mM = 0.013, p 100mM = 0.019, and p 150mM = 0.002). Apoptosis was significantly elevated, but the proportion of RGCs with neurite extension was significantly lower in the 50 mM, 100 mM, and 150 mM groups (p 50mM < 0.001, p 100mM < 0.001, and p 150mM < 0.001). CONCLUSIONS: We have optimized glucose concentrations to model diabetic retinal endothelial (25-50 mM) or neuronal (50-100 mM) dysfunction in vitro, which have a wide range of downstream applications.

Systemic treatment with 7,8-Dihydroxiflavone activates TtkB and affords protection of two different retinal ganglion cell populations against axotomy in adult rats.

PURPOSE: To analyze responses of different RGC populations to left intraorbital optic nerve transection (IONT) and intraperitoneal (i.p.) treatment with 7,8-Dihydroxyflavone (DHF), a potent selective TrkB agonist. METHODS: Adult albino Sprague-Dawley rats received, following IONT, daily i.p. injections of vehicle (1%DMSO in 0.9%NaCl) or DHF. Group-1 (n = 58) assessed at 7days (d) the optimal DHF amount (1-25 mg/kg). Group-2, using freshly dissected naïve or treated retinas (n = 28), investigated if DHF treatment was associated with TrkB activation using Western-blotting at 1, 3 or 7d. Group-3 (n = 98) explored persistence of protection and was analyzed at survival intervals from 7 to 60d after IONT. Groups 2-3 received daily i.p. vehicle or DHF (5 mg/kg). Retinal wholemounts were immunolabelled for Brn3a and melanopsin to identify Brn3a+RGCs and m+RGCs, respectively. RESULTS: Optimal neuroprotection was achieved with 5 mg/kg DHF and resulted in TrkB phosphorylation. The percentage of surviving Brn3a+RGCs in vehicle treated rats was 60, 28, 18, 13, 12 or 8% of the original value at 7, 10, 14, 21, 30 or 60d, respectively, while in DHF treated retinas was 94, 70, 64, 17, 10 or 9% at the same time intervals. The percentages of m+RGCs diminished by 7d-13%, and recovered by 14d-38% in vehicle-treated and to 48% in DHF-treated retinas, without further variations. CONCLUSIONS: DHF neuroprotects Brn3a + RGCs and m + RGCs; its protective effects for Brn3a+RGCs are maximal at 7 days but still significant at 21d, whereas for m+RGCs neuroprotection was significant at 14d and permanent.

Involvement of c-Jun N-terminal kinase 2 (JNK2) in Endothelin-1 (ET-1) Mediated Neurodegeneration of Retinal Ganglion Cells.

Purpose: The goal of this study was to determine whether JNK2 played a causative role in endothelin-mediated loss of RGCs in mice. Methods: JNK2-/- and wild type (C57BL/6) mice were intravitreally injected in one eye with 1 nmole of ET-1, whereas the contralateral eye was injected with the vehicle. At two time points (two hours and 24 hours) after the intravitreal injections, mice were euthanized, and phosphorylated c-Jun was assessed in retinal sections. In a separate set of experiments, JNK2-/- and wild type mice were intravitreally injected with either 1 nmole of ET-1 or its vehicle and euthanized seven days after injection. Retinal flat mounts were stained with antibodies to the RGC marker, Brn3a, and surviving RGCs were quantified. Axonal degeneration was assessed in paraphenylenediamine stained optic nerve sections. Results: Intravitreal ET-1 administration produced a significant increase in immunostaining for phospho c-Jun in wild type mice, which was appreciably lower in the JNK2 -/- mice. A significant (P < 0.05) 26% loss of RGCs was found in wild type mice, seven days after injection with ET-1. JNK2-/- mice showed a significant protection from RGC loss following ET-1 administration, compared to wild type mice injected with ET-1. A significant decrease in axonal counts and an increase in the collapsed axons was found in ET-1 injected wild type mice eyes. Conclusions: JNK2 appears to play a major role in ET-1 mediated loss of RGCs in mice. Neuroprotective effects in JNK2-/- mice following ET-1 administration occur mainly in the soma and not in the axons of RGCs.

Progressive Loss of Retinal Ganglion Cells in Activating Protein-2ß Neural Crest Cell Knockout Mice.

Purpose: Our lab has shown that conditionally disrupting the transcription factor activating protein 2ß (Tfap2b) gene, responsible for the activating protein-2ß (AP-2ß) transcription factor, exclusively in cranial neural crest cells (AP-2ß NCC KO), leads to anterior segment dysgenesis and a closed angle phenotype. The purpose of the current study is to determine if there is a progressive loss of retinal ganglion cells (RGCs) in the mutant over time and whether this loss was associated with macroglial activity changes and elevated intraocular pressure (IOP).Methods: Using the Cre-loxP system, we generated a conditional knockout of Tfap2b exclusively in cranial NCC (AP-2ß NCC KO). Immunohistochemistry was performed using anti-Brn3a, anti-GFAP and anti-Vimentin antibodies. IOP was measured using a tonometer and the data was analyzed using GraphPad Prism software. Brn3a and DAPI positive cells were counted using Image-J and statistical analysis was performed with GraphPad Prism software.Results: Our findings revealed that while no statistical difference in Brn3a expression was observed between wild-type and mutant mice at postnatal day (P) 4 or P10, at P40 (p < .01) and P42 (p < .0001) Brn3a expression was significantly reduced in the mutant retina at the region of the ONH. There was also increased expression of glial fibrillary acidic protein (GFAP) by Müller cells in the AP-2ß NCC KO mice at P35 and P40, indicating the presence of neuroinflammation. Moreover, increased IOP was observed starting at P35 and continuing at P40 and P42 (p < .0001 for all three ages examined).Conclusions: Together, these findings suggest that the retinal damage observed in the KO mouse becomes apparent by P40 after increased IOP was observed at P35 and progressed over time. The AP-2ß NCC KO mouse may therefore be a novel experimental model for glaucoma.

Biomarkers of lesion severity in a rodent model of nonarteritic anterior ischemic optic neuropathy (rNAION).

The rodent model of nonarteritic anterior ischemic optic neuropathy (rNAION) is similar in many of its pathophysiological responses to clinical NAION. Like human NAION, there is significant variability in the severity of the lesion produced, and little is known of the parameters associated with rNAION induction severity or if pre- or early post-induction biomarkers can be identified that enable prediction of lesion severity and ultimate loss of retinal ganglion cells (RGCs). Adult male Sprague-Dawley outbred rats were evaluated for various parameters including physiological characteristics (heart rate, respiratory rate, temperature, hematocrit [Hct]), optic nerve head (ONH) appearance, pre- and post-induction mean diameter, and intravenous fluorescein and indocyanine green angiographic patterns of vascular leakage at 5 hours post-induction, performed using a spectral domain-optical coherence tomography (SD-OCT) instrument. Early changes were correlated with ultimate RGC loss by Brn3a (+) immunohistology. RGC loss also was correlated with the relative level of laser exposure. The severity of ONH edema 2d, but not 5hr, post induction was most closely associated with the degree of RGC loss, revealing a threshold effect, and consistent with a compartment syndrome where a minimum level of capillary compression within a tight space is responsible for damage. RGC loss increased dramatically as the degree of laser exposure increased. Neither physiological parameters nor the degree of capillary leakage 5hr post induction were informative as to the ultimate degree of RGC loss. Similar to human NAION, the rNAION model exhibits marked variability in lesion severity. Unlike clinical NAION, pre-induction ONH diameter likely does not contribute to ultimate lesion severity; however, cross-sectional ONH edema can be used as a biomarker 2d post-induction to determine randomization of subjects prior to inclusion in specific neuroprotection or neuroregeneration studies.

Dual Specific Phosphatase 14 Deletion Rescues Retinal Ganglion Cells and Optic Nerve Axons after Experimental Anterior Ischemic Optic Neuropathy.

PURPOSE: Understanding molecular changes is essential for designing effective treatments for nonarteritic anterior ischemic optic neuropathy (AION), the most common acute optic neuropathy in adults older than 50 years. We investigated changes in the mitogen-activated protein kinase (MAPK) pathway after experimental AION and focused on dual specificity phosphatase 14 (Dusp14), an atypical MAPK phosphatase that is downstream of Krüppel-like transcription factor (KLF) 9-mediated inhibition of retinal ganglion cell (RGC) survival and axonal regeneration. MATERIALS AND METHODS: We induced severe AION in a photochemical thrombosis model in adult C57BL/6 wild-type and Dusp14 knockout mice. For comparison, some studies were performed using an optic nerve crush model. We assessed changes in MAPK pathway molecules using Western blot and immunohistochemistry, measured retinal thickness using optical coherence tomography (OCT), and quantified RGCs and axons using histologic methods. RESULTS: Three days after severe AION, there was no change in the retinal protein levels of MAPK ERK1/2, phosphorylated-ERK1/2 (pERK1/2), downstream effector Elk-1 and phosphatase Dusp14 on Western blot. Western blot analysis of purified RGCs after a more severe model using optic nerve crush also showed no change in Dusp14 protein expression. Because of the known importance of the Dusp14 and MAPK pathway in RGCs, we examined changes after AION in Dusp14 knockout mice. Three days after AION, Dusp14 knockout mice had significantly increased pERK1/2+, Brn3A+ RGCs on immunohistochemistry. Three weeks after AION, Dusp14 knockout mice had significantly greater preservation of retinal thickness, increased number of Brn3A+ RGCs on whole mount preparation, and increased number of optic nerve axons compared with wild-type mice. CONCLUSIONS: Genetic deletion of Dusp14, a MAPK phosphatase important in KFL9-mediated inhibition of RGC survival, led to increased activation of MAPK ERK1/2 and greater RGC and axonal survival after experimental AION. Inhibiting Dusp14 or activating the MAPK pathway should be examined further as a potential therapeutic approach to treatment of AION.Abbreviations: AION: anterior ischemic optic neuropathy; Dusp14: dual specific phosphatase 14; ERK1/2: extracellular signal-regulated kinases 1/2; Elk-1: ETS Like-1 protein; GCC: ganglion cell complex; GCL: ganglion cell layer; inner nuclear layer; KO: knockout; MAPK: mitogen-activated phosphokinase; OCT: optical coherence tomography; RGC: retinal ganglion cell; RNFL: retinal nerve fiber layer.

Overexpression of D-amino acid oxidase prevents retinal neurovascular pathologies in diabetic rats.

AIMS/HYPOTHESIS: Diabetic retinopathy is characterised by retinal neurodegeneration and retinal vascular abnormalities, affecting one third of diabetic patients with disease duration of more than 10 years. Accumulated evidence suggests that serine racemase (SR) and D-serine are correlated with the pathogenesis of diabetic retinopathy and the deletion of the Srr gene reverses neurovascular pathologies in diabetic mice. Since D-serine content is balanced by SR synthesis and D-amino acid oxidase (DAAO) degradation, we examined the roles of DAAO in diabetic retinopathy and further explored relevant therapy. METHODS: Rats were used as a model of diabetes by i.p. injection of streptozotocin at the age of 2 months and blood glucose was monitored with a glucometer. Quantitative real-time PCR was used to examine Dao mRNA and western blotting to examine targeted proteins in the retinas. Bisulphite sequencing was used to examine the methylation of Dao mRNA promoter in the retinas. Intravitreal injection of DAAO-expressing adenovirus (AAV8-DAAO) was conducted one week before streptozotocin administration. Brain specific homeobox/POU domain protein 3a (Brn3a) immunofluorescence was conducted to indicate retinal ganglion cells at 3 months after virus injection. The permeability of the blood-retinal barrier was examined by Evans blue leakage from retinal capillaries. Periodic acid-Schiff staining and haematoxylin counterstaining were used to indicate retinal vasculature, which was further examined with double immunostaining at 7 months after virus injection. RESULTS: At the age of 12 months, DAAO mRNA and protein levels in retinas from diabetic animals were reduced to 66.2% and 70.4% of those from normal (control) animals, respectively. The Dao proximal promoter contained higher levels of methylation in diabetic than in normal retinas. Consistent with the observation, DNA methyltransferase 1 was increased in diabetic retinas. Injection of DAAO-expressing virus completely prevented the loss of retinal ganglion cells and the disruption of blood-retinal barrier in diabetic rats. Diabetic retinas contained retinal ganglion cells at a density of 54 ± 4/mm2, which was restored to 68 ± 9/mm2 by DAAO overexpression, similar to the levels in normal retinas. The ratio between the number of endothelial cells and pericytes in diabetic retinas was 6.06 ± 1.93/mm2, which was reduced to 3.42 ± 0.55/mm2 by DAAO overexpression; the number of acellular capillaries in diabetic retinas was 10 ± 5/mm2, which was restored to 6 ± 2/mm2 by DAAO overexpression, similar to the levels in normal retinas. Injection of the DAAO-expressing virus increased the expression of occludin and reduced gliosis, which were examined to probe the mechanism by which the disrupted blood-retinal barrier in diabetic rats was rescued and retinal neurodegeneration was prevented. CONCLUSIONS/INTERPRETATION: Altogether, overexpression of DAAO before the onset of diabetes protects against neurovascular abnormalities in retinas from diabetic rats, which suggests a novel strategy for preventing diabetic retinopathy. Graphical abstract.

Sex Does Not Influence Visual Outcomes After Blast-Mediated Traumatic Brain Injury but IL-1 Pathway Mutations Confer Partial Rescue.

Purpose: In a mouse model of blast-mediated traumatic brain injury (bTBI), interleukin-1 (IL-1)-pathway components were tested as potential therapeutic targets for bTBI-mediated retinal ganglion cell (RGC) dysfunction. Sex was also evaluated as a variable for RGC outcomes post-bTBI. Methods: Male and female mice with null mutations in genes encoding IL-1α, IL-1ß, or IL-1RI were compared to C57BL/6J wild-type (WT) mice after exposure to three 20-psi blast waves given at an interblast interval of 1 hour or to mice receiving sham injury. To determine if genetic blockade of IL-1α, IL-1ß, or IL-1RI could prevent damage to RGCs, the function and structure of these cells were evaluated by pattern electroretinogram and optical coherence tomography, respectively, 5 weeks following blast or sham exposure. RGC survival was also quantitatively assessed via immunohistochemical staining of BRN3A at the completion of the study. Results: Our results showed that male and female WT mice had a similar response to blast-induced retinal injury. Generally, constitutive deletion of IL-1α, IL-1ß, or IL-1RI did not provide full protection from the effects of bTBI on visual outcomes; however, injured WT mice had significantly worse visual outcomes compared to the injured genetic knockout mice. Conclusions: Sex does not affect RGC outcomes after bTBI. The genetic studies suggest that deletion of these IL-1 pathway components confers some protection, but global deletion from birth did not result in a complete rescue.

POU4F1 confers trastuzumab resistance in HER2-positive breast cancer through regulating ERK1/2 signaling pathway.

Over-expression of the human epidermal growth factor receptor-2 (HER2) is related to aggressive tumors and poor prognosis in breast cancer. Trastuzumab (TRA) resistance leads to tumor recurrence and metastasis, resulting in poor prognosis in HER2-positive breast cancer. POU Class 4 Homeobox 1 (POU4F1) is a member of the POU domain family transcription factors, and has a key role in regulating cancers. However, its effects on TRA-resistant HER2-positive breast cancer are still vague. In the present study, we found that POU4F1 expression was dramatically increased in clinical breast cancer specimens with TRA resistance. Higher POU4F1 was also detected in HER2-positive breast cancer cells with TRA resistance than that of the parental ones. Poor prognosis was detected in breast cancer patients with high POU4F1 expression. Under TRA treatment, POU4F1 knockdown significantly reduced the proliferative capacity of HER2-positive breast cancer cells with TRA resistance. POU4F1 silence also sensitized resistant HER-positive breast cancer cells to TRA treatment in vivo using a xenograft mouse model, along with the markedly reduced tumor growth rate and tumor weight. Moreover, we found that POU4F1 deletion greatly decreased the activation of mitogen-activated or extracellular signal-regulated protein kinase kinases 1 and 2 (MEK1/2) and extracellular-regulated kinase 1/2 (ERK1/2) signaling pathways in breast cancer cells with TRA resistance. Migration and invasion were also effectively hindered by POU4F1 knockdown in TRA-resistant HER2-positive breast cancer cells. Notably, we found that POU4F1 deletion-improved chemosensitivity of HER2-positive breast cancer cells with drug-resistance to TRA treatment was closely associated with the blockage of ERK1/2 signaling. Collectively, our findings reported a critical role of POU4F1 in regulating TRA resistance, and demonstrated the underlying molecular mechanisms in HER2-positive breast cancer. Thus, POU4F1 may be a promising prognostic and therapeutic target to develop effective treatment for overcoming TRA resistance.

GFI1 functions to repress neuronal gene expression in the developing inner ear hair cells.

Despite the known importance of the transcription factors ATOH1, POU4F3 and GFI1 in hair cell development and regeneration, their downstream transcriptional cascades in the inner ear remain largely unknown. Here, we have used Gfi1cre;RiboTag mice to evaluate changes to the hair cell translatome in the absence of GFI1. We identify a systematic downregulation of hair cell differentiation genes, concomitant with robust upregulation of neuronal genes in the GFI1-deficient hair cells. This includes increased expression of neuronal-associated transcription factors (e.g. Pou4f1) as well as transcription factors that serve dual roles in hair cell and neuronal development (e.g. Neurod1, Atoh1 and Insm1). We further show that the upregulated genes are consistent with the NEUROD1 regulon and are normally expressed in hair cells prior to GFI1 onset. Additionally, minimal overlap of differentially expressed genes in auditory and vestibular hair cells suggests that GFI1 serves different roles in these systems. From these data, we propose a dual mechanism for GFI1 in promoting hair cell development, consisting of repression of neuronal-associated genes as well as activation of hair cell-specific genes required for normal functional maturation.

Neural transcription factor Pou4f1 promotes renal fibrosis via macrophage-myofibroblast transition.

Unresolved inflammation can lead to tissue fibrosis and impaired organ function. Macrophage-myofibroblast transition (MMT) is one newly identified mechanism by which ongoing chronic inflammation causes progressive fibrosis in different forms of kidney disease. However, the mechanisms underlying MMT are still largely unknown. Here, we discovered a brain-specific homeobox/POU domain protein Pou4f1 (Brn3a) as a specific regulator of MMT. Interestingly, we found that Pou4f1 is highly expressed by macrophages undergoing MMT in sites of fibrosis in human and experimental kidney disease, identified by coexpression of the myofibroblast marker, α-SMA. Unexpectedly, Pou4f1 expression peaked in the early stage in renal fibrogenesis in vivo and during MMT of bone marrow-derived macrophages (BMDMs) in vitro. Mechanistically, chromatin immunoprecipitation (ChIP) assay identified that Pou4f1 is a Smad3 target and the key downstream regulator of MMT, while microarray analysis defined a Pou4f1-dependent fibrogenic gene network for promoting TGF-ß1/Smad3-driven MMT in BMDMs at the transcriptional level. More importantly, using two mouse models of progressive renal interstitial fibrosis featuring the MMT process, we demonstrated that adoptive transfer of TGF-ß1-stimulated BMDMs restored both MMT and renal fibrosis in macrophage-depleted mice, which was prevented by silencing Pou4f1 in transferred BMDMs. These findings establish a role for Pou4f1 in MMT and renal fibrosis and suggest that Pou4f1 may be a therapeutic target for chronic kidney disease with progressive renal fibrosis.

Toll-like receptor-9 (TLR-9) deficiency alleviates optic nerve injury (ONI) by inhibiting inflammatory response in vivo and in vitro.

Traumatic optic neuropathy is a common clinical problem. Damage to the optic nerve leads to shear stress and triggers secondary swelling within the optic canal. The study aims to explore the role of the inflammatory response following optic nerve injury (ONI) in toll-like receptor-9 knockout mice (TLR-9-/-) compared to wild-type mice (WT). At first, TLR-9-/- and WT mice were subjected to ONI. We then found that ONI significantly up-regulated TLR-9 expression levels in retinal tissues of WT mice. The retinal degeneration after ONI was alleviated in TLR-9-/- mice, as evidenced by the increased number of retinal ganglion cells (RGCs) and thickness of inner retinal layer (IRL). TUNEL staining and immunofluorescence staining of BRN3A indicated that TLR-9 knockout effectively improved the survival of RGCs. ONI-enhanced expression of Iba-1 and TMEM119 was markedly reduced in TLR-9-/- mice, indicating the suppression of microglial activation. Moreover, production of pro-inflammatory regulators, including inducible nitric oxide synthase (iNOS), macrophage chemo-attractant protein (MCP)-1, cyclooxygenase-2 (COX-2), interleukin (IL)-1ß, IL-18 and tumor necrosis factor-α (TNF-α), was significantly decreased in TLR-9-/- mice following ONI. TLR-9 knockout-attenuated inflammation was mainly through repressing myeloid differentiation factor 88 (MyD88) and IL-1 receptor-associated kinase 4 (IRAK4). Furthermore, ONI greatly up-regulated the protein expression levels of phosphorylated (p)-IKKα, p-IκBα and p-nuclear factor (NF)-κB, whereas being repressed in TLR-9-/- mice. The effects of TLR-9 on ONI were verified in lipopolysaccharide (LPS)-stimulated retinal microglial cells transfected with small interfering RNA TLR-9 (siTLR-9). As expected, promoting TLR-9 with its agonist markedly restored inflammation in TLR-9 knockdown cells stimulated by LPS. Therefore, all findings above suggested that suppressing TLR-9 showed neuroprotective effects against ONI through reducing inflammatory response, and TILR-9 might be a promising therapeutic target to develop effective strategies for the treatment of optic neuropathies.

Transient receptor potential vanilloid type 1 is expressed in the horizontal pathway of the vervet monkey retina.

The ubiquitous distribution of the classic endocannabinoid system (cannabinoid receptors CB1 and CB2) has been demonstrated within the monkey nervous system, including the retina. Transient receptor potential vanilloid type 1 (TRPV1) is a cannabinoid-like non-selective cation channel receptor that is present in the retina and binds to endovannilloids and endocannabinoids, like anandamide, 2-arachidonoylglycerol and N-arachidonoyl dopamine. Retinal expression patterns of TRPV1 are available for rodents and data in higher mammals like humans and monkeys are scarce. We therefore thoroughly examined the expression and localization of TRPV1 in the retina, at various eccentricities, of the vervet (Chlorocebus sabeus) monkey, using Western blots and immunohistochemistry. Our results demonstrate that TRPV1 is found mainly in the outer and inner plexiform layers, and in the retinal ganglion cell (RGC) layer with a higher density in the periphery. Co-immunolabeling of TRPV1 with parvalbumin, a primate horizontal cell marker, revealed a clear overlap of expression throughout the entire cell structure with most prominent staining in the cell body membrane and synaptic terminals. Furthermore, double labeling of TRPV1 and syntaxin was found throughout amacrine cells in the inner plexiform layer. Finally, double staining of TRPV1 and Brn3a allowed us to confirm its previously reported expression in the cell bodies and dendrites of RGCs. The presence of TRPV1 in the horizontal pathway suggests a function of this receptor in lateral inhibition between photoreceptors through the horizontal cells, and between bipolar cells through amacrine cells.

POU4F1 promotes the resistance of melanoma to BRAF inhibitors through MEK/ERK pathway activation and MITF up-regulation.

BRAF inhibitors (BRAFi) have shown remarkable clinical efficacy in the treatment of melanoma with BRAF mutation. Nevertheless, most patients end up with the development of BRAFi resistance, which strongly limits the clinical application of these agents. POU4F1 is a stem cell-associated transcriptional factor that is highly expressed in melanoma cells and contributes to BRAF-activated malignant transformation. However, whether POU4F1 contributes to the resistance of melanoma to BRAFi remains poorly understood. Here, we report that over-expressed POU4F1 contributed to the acquired resistance of melanoma cells to Vemurafenib. Furthermore, POU4F1 promoted the activation of ERK signaling pathway via transcriptional regulation on MEK expression. In addition, POU4F1 could increase the expression of MITF to retain the resistance of melanoma cells to BRAFi. Collectively, our findings reveal that POU4F1 re-activates the MAPK pathway by transcriptional regulation on MEK expression and promotes MITF expression, which ultimately results in the resistance to BRAFi in melanoma. Our study supports that POU4F1 is a potential combined therapeutic target with BRAFi therapy for melanoma.

Progesterone Receptor Expression in the Benign Prostatic Hyperplasia and Prostate Cancer Tissues, Relation with Transcription, Growth Factors, Hormone Reception and Components of the AKT/mTOR Signaling Pathway.

BACKGROUND: Progesterone receptor (PR) is a critical regulator in reproductive tissues that controls a variety of cellular processes. The objective of the study was to study the PR expression in patients with benign prostatic hyperplasia and prostate cancers in connection with the transcription, growth factors, AR, ERα, ERß, and components of the AKT/mTOR signaling pathway expression. MATERIALS AND METHODS: Ninety-seven patients with prostate pathology were enrolled in the study. Forty-two patients had benign prostatic hyperplasia (BH). Fifty-five patients had locally advanced prostate cancer (PCa). The PSA level and the amount of testosterone in the serum were measured using an ELISA assay. The expression level of NF-κB p65, NF-κB p50, HIF-1, HIF-2, growth factor VEGF, VEGFR2, CAIX, as well as AR, ERα, ERß, PR, Brn-3α, TRIM16 were quantified by RT-PCR. The protein level of Brn-3α, TRIM16 was detected by Western Blotting. RESULTS: Growth in PR expression was observed in PCa tissues compared to BH ones without changes in the clinical and pathological features of the patients. An increase in PR expression was detected in patients with PCa compared to BH. Its mRNA level depended on the expression of AR, Brn-3α, and TRIM16, components of the AKT/mTOR signaling pathway, transcription, and growth factors. An increase in the TRIM16 expression in the PCa tissues was noted in the case of a low PR level. We revealed the growth in PR expression was accompanied by the suppression of the signaling cascade activity, AR, Brn-3α mRNA level, and the enhanced PTEN expression in PCa tissues. The increase in PR expression in PCa led to a decrease in the level of mRNA of NF-κB, HIF-1, VEGF, and VEGFR2. CONCLUSION: In general, the data indicated the significance of the PR expression in the development of the prostate pathology that affected the cross-talk between the steroid hormone reception and signal transduction. 
.

Transcriptional Programming of Human Mechanosensory Neuron Subtypes from Pluripotent Stem Cells.

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 overexpressing 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.

Mitochondria and Autophagy Dysfunction in Glucocorticoid-Induced Ocular Hypertension/Glaucoma Mice Model.

Purpose: This study is aimed to investigate the effects of periocular steroids induction on intraocular pressure (IOP), retinal ganglion cells (RGCs) and trabecular meshwork (TM) ultrastructure in glucocorticoid-induced ocular hypertension mice model.Materials and Methods: Dexamethasone-21-acetate (Dex-Ace) was administered through periocular conjunctival fornix injection every 3 days in C57BL/6J mice. Intraocular pressure was measured weekly by rebound tonometry. RGCs were examined with immunofluorescent staining of BRN3a at week 1, 4, and 8. TM morphology was visualized with electron microscopy. Autophagy was evaluated with immunoblotting in TM tissues.Results: Dex-Ace rapidly and significantly induced IOP, which peaked at week 4. The absolute increase in IOP in the Dex-Ace-treated mice was 8.1 ± 1.4 mmHg, a 60% induction (p < .0001) compared with that in the vehicle-treated mice. The IOP sustained a higher level in the Dex-Ace group from week 4 to week 8. Dex-Ace treatment decreased the number of RGCs in a time-dependent manner, suggesting that high IOP resulted in optic neuropathy. In addition, Dex-Ace thickened trabecular beams and decreased intertrabecular spaces, with marked accumulation of fibrillar and amorphous granular extracellular material. Moreover, Dex-Ace induced swollen and elongated mitochondria in TM cells. The average mitochondria area was 0.090 ± 0.044 µm2 in the vehicle-treated mice, and increased to 0.161 ± 0.094 µm2 (p < .0001), 0.121 ± 0.029 µm2 (p = .0223) and 0.171 ± 0.076 µm2 (p < .0001) in the Dex-Ace-treated mice at weeks 1, 4 and 8, respectively. Autophagy was also increased by Dex-Ace treatment, indicating by the upregulation of LC3-I, LC3-II and beclin-1, and downregulation of p62.Conclusion: Dex-Ace administration decreased RGCs and changed TM ultrastructure, mimicking hallmarks of human glucocorticoid-induced glaucoma (GIG). In addition, mitochondria and autophagy dysfunction suggested abnormal energy metabolism in TM cells, which warranted further study to fully elucidate the pathogenesis of GIG.