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Superior segmental optic nerve hypoplasia (SSONH) is a congenital condition characterized by developmental abnormalities of the superior optic disc and an underappreciated differential diagnosis for glaucoma. The reported prevalence is less than 1%, although likely underestimated due to the difficulties with diagnosis. The exact pathophysiology of SSONH remains elusive, but a mechanism involving developmental attrition of retinal ganglion cells has been proposed, and maternal diabetes is recognized as a major risk factor. SSONH often is observed incidentally, and the patients typically are then evaluated for an acquired optic atrophy, often glaucoma because of the presence of inferior visual field defects. There are 4 characteristic signs of SSONH: superior entrance of the central retinal artery, superior disc pallor, superior peripapillary halo, and thinning of the superior peripapillary nerve fiber layer; however, the presence of these signs is variable. Optical coherence tomography can be helpful in distinguishing SSONH by demonstrating superonasal retinal nerve fiber layer thinning, as compared to the inferotemporal thinning seen in glaucoma, and an aberrant extension of retinal pigment epithelium over Bruch membrane. Overall, the prognosis of SSONH is favorable, with a non-progressive course. It is essential that ophthalmologists recognize and differentiate SSONH from glaucoma to avoid misdiagnosis and unnecessary treatment.

Transcriptional activity of the androgen receptor (AR) is crucial for growth and survival of prostate cancer even upon development of resistance to androgen ablation and antiandrogen therapies. Therefore, novel therapies that can suppress AR transcriptional activity when conventional hormone therapies fail are needed. Here, we show that histone deacetylase (HDAC) inhibitors, including SAHA (vorinostat) and LBH589, which are currently being tested in clinic, could be such a therapy. HDAC inhibitors block the AR-mediated transcriptional activation of many genes, including the TMPRSS2 gene involved in fusion with ETS family members in a majority of prostate cancers. Genetic knockdown of either HDAC1 or HDAC3 can also suppress expression of AR-regulated genes, recapitulating the effect of HDAC inhibitor treatment. Whereas HDAC inhibitor treatment can lower androgen receptor protein levels in prostate cancer cells, we show that independent of AR protein levels, HDAC inhibitors block AR activity through inhibiting the assembly of coactivator/RNA polymerase II complex after AR binds to the enhancers of target genes. Failed complex assembly is associated with a phase shift in the cyclical wave of AR recruitment that typically occurs in response to ligand treatment. HDAC inhibitors retain the ability to block AR activity in castration-resistant prostate cancer models and, therefore, merit clinical investigation in this setting. The HDAC-regulated AR target genes defined here can serve as biomarkers to ensure sufficient levels of HDAC inhibition. [Cancer Res 2009;69(3):958–66]

Supplementary Figure 3 from Histone Deacetylases Are Required for Androgen Receptor Function in Hormone-Sensitive and Castrate-Resistant Prostate Cancer

Supplementary Figure Legends 1-4 from Histone Deacetylases Are Required for Androgen Receptor Function in Hormone-Sensitive and Castrate-Resistant Prostate Cancer

Supplementary Figure 1 from Histone Deacetylases Are Required for Androgen Receptor Function in Hormone-Sensitive and Castrate-Resistant Prostate Cancer

Supplementary Table 1 from Histone Deacetylases Are Required for Androgen Receptor Function in Hormone-Sensitive and Castrate-Resistant Prostate Cancer

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Significance Axonal injury plays a major role in many neurodegenerative diseases. The dual leucine zipper kinase (DLK) signaling pathway is a key regulator of axonal injury-induced neuronal cell death; however, DLK also has an important role in promoting axonal outgrowth. Therefore, inhibiting DLK as a therapeutic approach for neurodegenerative diseases is limited to a neuroprotective outcome without axon regeneration, prohibiting restoration of function. In fact, there are currently no strategies that provide long-term neuroprotection and axonal regeneration after injury. Here, we identified the germinal cell kinase four (GCK-IV) family of kinases as targets to maximize neuroprotection while promoting axon regeneration, making it an attractive therapeutic approach for a subset of neurodegenerative diseases., Axon injury is a hallmark of many neurodegenerative diseases, often resulting in neuronal cell death and functional impairment. Dual leucine zipper kinase (DLK) has emerged as a key mediator of this process. However, while DLK inhibition is robustly protective in a wide range of neurodegenerative disease models, it also inhibits axonal regeneration. Indeed, there are no genetic perturbations that are known to both improve long-term survival and promote regeneration. To identify such a neuroprotective target, we conducted a set of complementary high-throughput screens using a protein kinase inhibitor library in human stem cell-derived retinal ganglion cells (hRGCs). Overlapping compounds that promoted both neuroprotection and neurite outgrowth were bioinformatically deconvoluted to identify specific kinases that regulated neuronal death and axon regeneration. This work identified the role of germinal cell kinase four (GCK-IV) kinases in cell death and additionally revealed their unexpected activity in suppressing axon regeneration. Using an adeno-associated virus (AAV) approach, coupled with genome editing, we validated that GCK-IV kinase knockout improves neuronal survival, comparable to that of DLK knockout, while simultaneously promoting axon regeneration. Finally, we also found that GCK-IV kinase inhibition also prevented the attrition of RGCs in developing retinal organoid cultures without compromising axon outgrowth, addressing a major issue in the field of stem cell-derived retinas. Together, these results demonstrate a role for the GCK-IV kinases in dissociating the cell death and axonal outgrowth in neurons and their druggability provides for therapeutic options for neurodegenerative diseases.

To investigate the efficacy of a deep learning regression method to predict macula ganglion cell-inner plexiform layer (GCIPL) and optic nerve head (ONH) retinal nerve fiber layer (RNFL) thickness for use in glaucoma neuroprotection clinical trials.Cross-sectional study.Glaucoma patients with good quality macula and ONH scans enrolled in 2 longitudinal studies, the African Descent and Glaucoma Evaluation Study and the Diagnostic Innovations in Glaucoma Study.Spectralis macula posterior pole scans and ONH circle scans on 3327 pairs of GCIPL/RNFL scans from 1096 eyes (550 patients) were included. Participants were randomly distributed into a training and validation dataset (90%) and a test dataset (10%) by participant. Networks had access to GCIPL and RNFL data from one hemiretina of the probe eye and all data of the fellow eye. The models were then trained to predict the GCIPL or RNFL thickness of the remaining probe eye hemiretina.Mean absolute error (MAE) and squared Pearson correlation coefficient (rThe deep learning model was able to predict superior and inferior GCIPL thicknesses with a global rOur deep learning models were able to accurately estimate both macula GCIPL and ONH RNFL hemiretinal thickness. Using an internal control based on these model predictions may help reduce clinical trial sample size requirements and facilitate investigation of new glaucoma neuroprotection therapies.

Optical coherence tomography (OCT) has gained wide adoption in biological research and medical imaging due to its exceptional tissue penetration, 3D imaging speed, and rich contrast. However, OCT plays a relatively small role in molecular and cellular imaging due to the lack of suitable biomolecular contrast agents. In particular, while the green fluorescent protein has provided revolutionary capabilities to fluorescence microscopy by connecting it to cellular functions such as gene expression, no equivalent reporter gene is currently available for OCT. Here, we introduce gas vesicles, a class of naturally evolved gas-filled protein nanostructures, as genetically encodable OCT contrast agents. The differential refractive index of their gas compartments relative to surrounding aqueous tissue and their nanoscale motion enables gas vesicles to be detected by static and dynamic OCT. Furthermore, the OCT contrast of gas vesicles can be selectively erased in situ with ultrasound, allowing unambiguous assignment of their location. In addition, gas vesicle clustering modulates their temporal signal, enabling the design of dynamic biosensors. We demonstrate the use of gas vesicles as reporter genes in bacterial colonies and as purified contrast agents in vivo in the mouse retina. Our results expand the utility of OCT to image a wider variety of cellular and molecular processes.

PurposeTo examine whether glaucomatous central visual field abnormalities can be more effectively detected using a qualitative, expert evaluation of the 10-2 test compared with the topographically corresponding central 12 locations of the 24-2 test (C24-2).DesignCross-sectional study.MethodsEyes with a glaucomatous optic nerve appearance or ocular hypertension (n=523) and healthy eyes (n=107) were included as cases and control subjects, respectively. The 10-2 and C24-2 visual field results of all eyes were graded by 4 glaucoma specialists for the probability that central visual field abnormalities were present.ResultsThe sensitivity of the 10-2 and C24-2 tests for detecting the cases at 95% specificity were not significantly different (e.g., 32.2% and 31.4%, respectively, for grader 1, P=.87; all graders P ≥ .25). At 95% specificity, the pattern standard deviation values from these tests had a similar sensitivity to the qualitative evaluation for the C24-2 test for all graders (P ≥ .083), but it had a significantly higher sensitivity than the qualitative evaluation for the 10-2 test for 3 graders (P ≤ .016).ConclusionsThe similarity in performance of the 10-2 and C24-2 test suggests that the increased sampling density of the former does not significantly improve the detection of central visual field abnormalities, even when based on expert assessment. These findings should not be taken to mean that the 10-2 test is not useful, but it underscores the need for its utility to be clearly established before incorporating it as routine glaucoma standard of care.

PURPOSE: To evaluate the thinning of circumpapillary retinal nerve fiber layer (cpRNFL) and macular ganglion cell-inner plexiform layer (mGCIPL) in primary open angle glaucoma (POAG) eyes with and without history of disc hemorrhage (DH). DESIGN: Observational cohort study SUBJECTS: 39 eyes (34 subjects) with DH and 117 eyes (104 subjects) without DH from the Diagnostic Innovations in Glaucoma Study (DIGS) and the African Decent and Glaucoma Evaluation Study (ADAGES). METHODS: All participants had at least 1.5-years of follow-up with a minimum of 3 visits with bi-annual spectral-domain optic coherence tomography (SD-OCT) cpRNFL and mGCIPL thickness measurements and visual fields (VF). The rates of cpRNFL and mGCIPL thinning were calculated using mixed effects models. The dynamic range-based normalized rates of cpRNFL and mGCIPL were calculated and compared between the DH and non-DH groups. MAIN OUTCOME MEASURES: Rates of cpRNFL and mGCIPL thinning RESULTS: The rate of mGCIPL thinning was significantly faster in the DH group compared to the non-DH group (−0.62 vs. −0.38 μm/year, P=0.024). The rate of cpRNFL thinning in the DH quadrant and rate of mGCIPL thinning in inferotemporal sector in the DH group were faster than the corresponding regions in the non-DH group after adjusting for intraocular pressure (IOP) and race (−1.33 vs. −0.58 μm/year, P=0.053, and (−0.82 vs. −0.44 μm/year, P = 0.048, respectively). In the DH group, the percent rate of loss was significantly faster with mGCIPL than cpRNFL (−1.59 vs. −1.31 %/year, P=0.046). mGCIPL thinning rates were weakly associated with MD slope, VFI slope, and GPA (R(2) = 3.6%(P=0.058), R(2)=2.4%(P=0.096), and R(2)= 5.2%(P=0.073), respectively). The area under operating receiver curves for VF progression based on Guided Progression Analysis was 0.75 for mGCIPL and 0.56 for cpRNFL in the DH group. CONCLUSIONS: In our study, the rate of mGCIPL and cpRNFL thinning was faster in DH eyes than non-DH eyes. mGCIPL showed higher proportional rates of thinning and greater association with functional progression compared to cpRNFL. In addition to cpRNFL, clinicians should consider incorporating mGCIPL imaging to monitor glaucoma progression, especially in glaucoma eyes with DH.

Retinal ganglion cell (RGC) axons converge at the optic nerve head to convey visual information from the retina to the brain. Pathologies such as glaucoma, trauma, and ischemic optic neuropathies injure RGC axons, disrupt transmission of visual stimuli, and cause vision loss. Animal models simulating RGC axon injury include optic nerve crush and transection paradigms. Each of these models has inherent advantages and disadvantages. An optic nerve crush is generally less severe than a transection and can be used to assay axon regeneration across the lesion site. However, differences in crush force and duration can affect tissue responses, resulting in variable reproducibility and lesion completeness. With optic nerve transection, there is a severe and reproducible injury that completely lesions all axons. However, transecting the optic nerve dramatically alters the blood brain barrier by violating the optic nerve sheath, exposing the optic nerve to the peripheral environment. Moreover, regeneration beyond a transection site cannot be assessed without reapposing the cut nerve ends. Furthermore, distinct degenerative changes and cellular pathways are activated by either a crush or transection injury. The method described here incorporates the advantages of both optic nerve crush and transection models while mitigating the disadvantages. Hydrostatic pressure delivered into the optic nerve by microinjection completely transects the optic nerve while maintaining the integrity of the optic nerve sheath. The transected optic nerve ends are reapposed to allow for axon regeneration assays. A potential limitation of this method is the inability to visualize the complete transection, a potential source of variability. However, visual confirmation that the visible portion of the optic nerve has been transected is indicative of a complete optic nerve transection with 90-95% success. This method could be applied to assess axon regeneration promoting strategies in a transection model or investigate interventions that target the axonal compartments.

Purpose of review: to provide a prospective on the current mechanisms by which SARS-CoV-2 enters cells and replicates, and its implications for ocular transmission. The literature was analyzed to understand ocular transmission as well as molecular mechanisms by which SARS-CoV-2 enters cells and replicates. Analysis of gene expression profiles from available datasets, published immunohistochemistry, as well as current literature was reviewed, to assess the likelihood that ocular inoculation of SARS-CoV-2 results in systemic infection. Recent findings: The ocular surface and retina have the necessary proteins, Transmembrane Serine Protease 2 (TMPRSS2), CD147, Angiotensin-Converting Enzyme 2 (ACE2) and Cathepsin L (CTSL) necessary to be infected with SARS-CoV-2. In addition to direct ocular infection, virus carried by tears through the nasolacrimal duct to nasal epithelium represent a means of ocular inoculation. Summary: There is evidence that SARS-CoV-2 may either directly infect cells on the ocular surface, or virus can be carried by tears through the nasolacrimal duct to infect the nasal or gastrointestinal epithelium.

PurposeTo compare the efficacy of intraoperative scleral application with subconjunctival injection of mitomycin C (MMC) in trabeculectomy.DesignProspective, randomized, interventional study.MethodsThis study took place in a single clinical practice in an academic setting. Patients had medically uncontrolled glaucoma as indicated by high intraocular pressure (IOP), worsening visual field, or optic nerve head changes in whom primary trabeculectomy was indicated. Patients were older than 18 years with medically uncontrolled glaucoma and no history of incisional glaucoma surgery. Patients were randomized to MMC delivered by preoperative subconjunctival injection or by intraoperative direct scleral application using surgical sponges during trabeculectomy. Comprehensive eye examinations were conducted at 1day, 1week, 6weeks, 3months, and 6months postoperatively. Subconjunctival 5-fluorouracil injections were given postoperatively, as needed. The primary outcome was the proportion of patients who demonstrated IOP of

Human pluripotent stem cells have the potential to promote biological studies and accelerate drug discovery efforts by making possible direct experimentation on a variety of human cell types of interest. However, stem cell cultures are generally heterogeneous and efficient differentiation and purification protocols are often lacking. Here, we describe the generation of clustered regularly-interspaced short palindromic repeats(CRISPR)-Cas9 engineered reporter knock-in embryonic stem cell lines in which tdTomato and a unique cell-surface protein, THY1.2, are expressed under the control of the retinal ganglion cell (RGC)-enriched gene BRN3B. Using these reporter cell lines, we greatly improved adherent stem cell differentiation to the RGC lineage by optimizing a novel combination of small molecules and established an anti-THY1.2-based protocol that allows for large-scale RGC immunopurification. RNA-sequencing confirmed the similarity of the stem cell-derived RGCs to their endogenous human counterparts. Additionally, we developed an in vitro axonal injury model suitable for studying signaling pathways and mechanisms of human RGC cell death and for high-throughput screening for neuroprotective compounds. Using this system in combination with RNAi-based knockdown, we show that knockdown of dual leucine kinase (DLK) promotes survival of human RGCs, expanding to the human system prior reports that DLK inhibition is neuroprotective for murine RGCs. These improvements will facilitate the development and use of large-scale experimental paradigms that require numbers of pure RGCs that were not previously obtainable.

Background Traumatic brain injury (TBI) is a major cause of CNS neurodegeneration and has no disease-altering therapies. It is commonly associated with a specific type of biomechanical disruption of the axon called traumatic axonal injury (TAI), which often leads to axonal and sometimes perikaryal degeneration of CNS neurons. We have previously used genome-scale, arrayed RNA interference-based screens in primary mouse retinal ganglion cells (RGCs) to identify a pair of related kinases, dual leucine zipper kinase (DLK) and leucine zipper kinase (LZK) that are key mediators of cell death in response to simple axotomy. Moreover, we showed that DLK and LZK are the major upstream triggers for JUN N-terminal kinase (JNK) signaling following total axonal transection. However, the degree to which DLK/LZK are involved in TAI/TBI is unknown. Methods Here we used the impact acceleration (IA) model of diffuse TBI, which produces TAI in the visual system, and complementary genetic and pharmacologic approaches to disrupt DLK and LZK, and explored whether DLK and LZK play a role in RGC perikaryal and axonal degeneration in response to TAI. Results Our findings show that the IA model activates DLK/JNK/JUN signaling but, in contrast to axotomy, many RGCs are able to recover from the injury and terminate the activation of the pathway. Moreover, while DLK disruption is sufficient to suppress JUN phosphorylation, combined DLK and LZK inhibition is required to prevent RGC cell death. Finally, we show that the FDA-approved protein kinase inhibitor, sunitinib, which has activity against DLK and LZK, is able to produce similar increases in RGC survival. Conclusion The mitogen-activated kinase kinase kinases (MAP3Ks), DLK and LZK, participate in cell death signaling of CNS neurons in response to TBI. Moreover, sustained pharmacologic inhibition of DLK is neuroprotective, an effect creating an opportunity to potentially translate these findings to patients with TBI.

Background Glaucoma affects more than 70 million people worldwide, with about 10% being bilaterally blind, making it the leading cause of irreversible blindness globally. In patients with advanced glaucoma or those who have failed medical treatment without achieving adequate intraocular pressure (IOP) control, trabeculectomy (glaucoma filtration surgery where an ostium is created into the anterior chamber from underneath a partial thickness scleral flap to allow for aqueous flow out of the anterior chamber intointo the subconjunctival space forming a filtering bleb) and aqueous shunt surgery for more complex and refractory cases remain the mainstay therapies. Proliferation of fibrous tissue around an implanted aqueous shunt may block the diffusion of aqueous humour. Mitomycin C (MMC) is one of two commonly used adjunct antifibrotic agents used during aqueous shunt surgery to prevent proliferation of fibrous tissue. However, the effectiveness and safety of the use of intraoperative MMC during aqueous shunt surgery has not been established. Objectives To evaluate the effectiveness and safety of MMC versus no MMC used during aqueous shunt surgery for reducing IOP in primary and secondary glaucoma. Search methods We searched the Cochrane Central Register of Controlled Trials (CENTRAL) (which contains the Cochrane Eyes and Vision Trials Register) (2018, Issue 2); Ovid MEDLINE; Embase.com; PubMed; Latin American and Caribbean Health Sciences Literature Database (LILACS); ClinicalTrials.gov and the World Health Organization (WHO) International Clinical Trials Registry Platform (ICTRP). We did not use any date or language restrictions in the electronic search for trials. We last searched the electronic databases on 13 February 2018. Selection criteria We included randomized controlled trials (RCTs) in which one group of participants received MMC during aqueous shunt surgery and another group did not. We did not exclude studies based on outcomes. Data collection and analysis Two review authors independently reviewed titles and abstracts from the literature searches. We obtained full-text reports of potentially relevant studies and assessed them for inclusion. Two review authors independently extracted data related to study characteristics, risk of bias, and outcomes. We used standard methodological procedures expected by Cochrane. Main results We included five RCTs, with a total of 333 eyes with glaucoma randomized, and identified two ongoing trials. All included trials examined the effect of MMC versus no MMC when used during aqueous shunt surgery for glaucoma. The trials included participants with different types of uncontrolled glaucoma. One study was conducted in China, one in Saudi Arabia, two in the USA, and one study was a multicenter study conducted in Brazil, Canada, Scotland, and USA. We assessed all trials as having overall unclear risk of bias due to incomplete reporting of study methods and outcomes; two of the five trials were reported only as conference abstracts.None of the included trials reported mean decrease from baseline in IOP; however, all five trials reported mean IOP at 12 months post-surgery. At 12 months, the effect of MMC on mean IOP compared with no MMC was unclear based on a meta-analysis of trials (mean difference -0.12 mmHg, 95% CI -2.16 to 2.41; low-certainty evidence). Two trial did not report sufficient information to include in meta-analysis, but reported that mean IOP was lower in the MMC group compared with the no MMC group at 12 months.None of the included trials reported mean change from baseline in visual acuity; however, one trial reported lower mean LogMAR values (better vision) in the MMC group than in the no MMC group at 12 months post-surgery. None of the included studies reported the proportion of participants with stable best-corrected visual acuity. Three trials reported that loss of vision was not significantly different between groups (no data available for meta-analysis).None of the included studies reported the proportion of participants with a postoperative hypertensive phase, which is defined as IOP > 21 mmHg within 3 months after surgery. Two trials reported adverse events (choroidal effusion, corneal edema, flat anterior chamber, and retinal detachment); however, due to small numbers of events and sample sizes, no clear difference between MMC and placebo groups was observed. Authors' conclusions We found insufficient evidence in this review to suggest MMC provides any postoperative benefit for glaucoma patients who undergo aqueous shunt surgery. Data across all five included trials were sparse and the reporting of study methods required to assess bias was inadequate. Future RCTs of this intervention should report methods in sufficient detail to permit assessment of potential bias and estimate target sample sizes based on clinically meaningful effect sizes.

Optical coherence tomography (OCT) has gained wide adoption in biological and medical imaging due to its exceptional tissue penetration, 3D imaging speed and rich contrast. However, OCT plays a relatively small role in molecular and cellular imaging due to the lack of suitable biomolecular contrast agents. In particular, while the green fluorescent protein has provided revolutionary capabilities to fluorescence microscopy by connecting it to cellular functions such as gene expression, no equivalent reporter gene is currently available for OCT. Here we introduce gas vesicles, a unique class of naturally evolved gas-filled protein nanostructures, as the first genetically encodable OCT contrast agents. The differential refractive index of their gas compartments relative to surrounding aqueous tissue and their nanoscale motion enables gas vesicles to be detected by static and dynamic OCT at picomolar concentrations. Furthermore, the OCT contrast of gas vesicles can be selectively erasedin situwith ultrasound, allowing unambiguous assignment of their location. In addition, gas vesicle clustering modulates their temporal signal, enabling the design of dynamic biosensors. We demonstrate the use of gas vesicles as reporter genes in bacterial colonies and as purified contrast agentsin vivoin the mouse retina. Our results expand the utility of OCT as a unique photonic modality to image a wider variety of cellular and molecular processes.

INTRODUCTION: Cone photoreceptors in the human retina enable daytime, color, and high-acuity vision. The three subtypes of human cones are defined by the visual pigment that they express: blue-opsin (short wavelength; S), green-opsin (medium wavelength; M), or red-opsin (long wavelength; L). Mutations that affect opsin expression or function cause various forms of color blindness and retinal degeneration. RATIONALE: Our current understanding of the vertebrate eye has been derived primarily from the study of model organisms. We studied the human retina to understand the developmental mechanisms that generate the mosaic of mutually exclusive cone subtypes. Specification of human cones occurs in a two-step process. First, a decision occurs between S versus L/M cone fates. If the L/M fate is chosen, a subsequent choice is made between expression of L- or M-opsin. To determine the mechanism that controls the first decision between S and L/M cone fates, we studied human retinal organoids derived from stem cells. RESULTS: We found that human organoids and retinas have similar distributions, gene expression profiles, and morphologies of cone subtypes. During development, S cones are specified first, followed by L/M cones. This temporal switch from specification of S cones to generation of L/M cones is controlled by thyroid hormone (TH) signaling. In retinal organoids that lacked thyroid hormone receptor β (Thrβ), all cones developed into the S subtype. Thrβ binds with high affinity to triiodothyronine (T3), the more active form of TH, to regulate gene expression. We observed that addition of T3 early during development induced L/M fate in nearly all cones. Thus, TH signaling through Thrβ is necessary and sufficient to induce L/M cone fate and suppress S fate. TH exists largely in two states: thyroxine (T4), the most abundant circulating form of TH, and T3, which binds TH receptors with high affinity. We hypothesized that the retina itself could modulate TH levels to control subtype fates. We found that deiodinase 3 (DIO3), an enzyme that degrades both T3 and T4, was expressed early in organoid and retina development. Conversely, deiodinase 2 (DIO2), an enzyme that converts T4 to active T3, as well as TH carriers and transporters, were expressed later in development. Temporally dynamic expression of TH-degrading and -activating proteins supports a model in which the retina itself controls TH levels, ensuring low TH signaling early to specify S cones and high TH signaling later in development to produce L/M cones. CONCLUSION: Studies of model organisms and human epidemiology often generate hypotheses about human biology that cannot be studied in humans. Organoids provide a system to determine the mechanisms of human development, enabling direct testing of hypotheses in developing human tissue. Our studies identify temporal regulation of TH signaling as a mechanism that controls cone subtype specification in humans. Consistent with our findings, preterm human infants with low T3 and T4 have an increased incidence of color vision defects. Moreover, our identification of a mechanism that generates one cone subtype while suppressing the other, coupled with successful transplantation and incorporation of stem cell-derived photoreceptors in mice, suggests that the promise of therapies to treat human diseases such as color blindness, retinitis pigmentosa, and macular degeneration will be achieved in the near future. ■, Graphical Abstract Temporally regulated TH signaling specifies cone subtypes. (A) Embryonic stem cell-derived human retinal organoids [wild type (WT)] generate S and L/M cones. Blue, S-opsin; green, L/M-opsin. (B) Organoids that lack thyroid hormone receptor β (Thrβ KO) generate all S cones. (C) Early activation of TH signaling (WT + T3) specifies nearly all L/M cones. (D) TH-degrading enzymes (such as DIO3) expressed early in development lower TH and promote S fate, whereas TH-activating regulators (such as DIO2) expressed later promote L/M fate., Summary The mechanisms underlying specification of neuronal subtypes within the human nervous system are largely unknown. The blue (S), green (M), and red (L) cones of the retina enable high-acuity daytime and color vision. To determine the mechanism that controls S versus L/M fates, we studied the differentiation of human retinal organoids. Organoids and retinas have similar distributions, expression profiles, and morphologies of cone subtypes. S cones are specified first, followed by L/M cones, and thyroid hormone signaling controls this temporal switch. Dynamic expression of thyroid hormone–degrading and –activating proteins within the retina ensures low signaling early to specify S cones and high signaling late to produce L/M cones. This work establishes organoids as a model for determining mechanisms of human development with promising utility for therapeutics and vision repair.

Optic neuropathies such as glaucoma are characterized by the degeneration of retinal ganglion cells (RGCs) and the irreversible loss of vision. In these diseases, focal axon injury triggers a propagating axon degeneration and, eventually, cell death. Previous work by us and others identified dual leucine zipper kinase (DLK) and JUN N-terminal kinase (JNK) as key mediators of somal cell death signaling in RGCs following axonal injury. Moreover, others have shown that activation of the DLK/JNK pathway contributes to distal axonal degeneration in some neuronal subtypes and that this activation is dependent on the adaptor protein, sterile alpha and TIR motif containing 1 (SARM1). Given that SARM1 acts upstream of DLK/JNK signaling in axon degeneration, we tested whether SARM1 plays a similar role in RGC somal apoptosis in response to optic nerve injury. Using the mouse optic nerve crush (ONC) model, our results show that SARM1 is critical for RGC axonal degeneration and that axons rescued by SARM1 deficiency are electrophysiologically active. Genetic deletion of SARM1 did not, however, prevent DLK/JNK pathway activation in RGC somas nor did it prevent or delay RGC cell death. These results highlight the importance of SARM1 in RGC axon degeneration and suggest that somal activation of the DLK/JNK pathway is activated by an as-yet-unidentified SARM1-independent signal.

Dual leucine zipper kinase (DLK) has been implicated in cell death signaling secondary to axonal damage in retinal ganglion cells (RGCs) and other neurons. To better understand the pathway through which DLK acts, we developed enhanced functional genomic screens in primary RGCs, including use of arrayed, whole-genome, small interfering RNA libraries. Explaining why DLK inhibition is only partially protective, we identify leucine zipper kinase (LZK) as cooperating with DLK to activate downstream signaling and cell death in RGCs, including in a mouse model of optic nerve injury, and show that the same pathway is active in human stem cell-derived RGCs. Moreover, we identify four transcription factors (JUN), activating transcription factor 2 (ATF2), myocyte-specific enhancer factor 2A (MEF2A), and SRY-Box 11 (SOX11)) as being the major downstream mediators through which DLK/LZK activation leads to RGC cell death. Increased understanding of the DLK pathway has implications for understanding and treating neurodegenerative diseases.

Non-mammalian vertebrates have an intrinsically photosensitive iris and thus a local pupillary light reflex (PLR). In contrast, it is thought that the PLR in mammals generally requires neuronal circuitry connecting the eye and the brain. Here we report that an intrinsic component of the PLR is in fact widespread in nocturnal and crepuscular mammals. In mouse, this intrinsic PLR requires the visual pigment melanopsin; it also requires PLCβ4, a vertebrate homologue of the Drosophila Norp A phospholipase C which mediates rhabdomeric phototransduction. The Plcb4^(−/−) genotype, in addition to removing the intrinsic PLR, also essentially eliminates the intrinsic light response of the M1 subtype of melanopsin-expressing, intrinsically photosensitive retinal ganglion cells (M1-ipRGCs), which are by far the most photosensitive ipRGC subtype and also have the largest response to light. Ablating in mouse the expression of both TRPC6 and TRPC7, members of the TRP channel superfamily, also essentially eliminated the M1-ipRGC light response but the intrinsic PLR was not affected. Thus, melanopsin signalling exists in both iris and retina, involving a PLCβ4-mediated pathway that nonetheless diverges in the two locations.

Delayed revascularization of ischemic neural tissue is a major impediment to preservation of function in central nervous system (CNS) diseases including stroke and ischemic retinopathies. Therapeutic strategies allowing rapid revascularization are greatly needed to reduce ischemia-induced cellular damage and suppress harmful pathologic neovascularization. However, key mechanisms governing vascular recovery in ischemic CNS, including regulatory molecules governing the transition from tissue injury to tissue repair, are largely unknown. NF-E2-related factor 2 (Nrf2) is a major stress-response transcription factor well known for its cell-intrinsic cytoprotective function. However, its role in cell-cell crosstalk is less appreciated. Here we report that Nrf2 is highly activated in ischemic retina and promotes revascularization by modulating neurons in their paracrine regulation of endothelial cells. Global Nrf2 deficiency strongly suppresses retinal revascularization and increases pathologic neovascularization in a mouse model of ischemic retinopathy. Conditional knockout studies demonstrate a major role for neuronal Nrf2 in vascular regrowth into avascular retina. Deletion of neuronal Nrf2 results in semaphorin 6A (Sema6A) induction in hypoxic/ischemic retinal ganglion cells in a hypoxia-inducible factor-1 alpha (HIF-1α)-dependent fashion. Sema6A expression increases in avascular inner retina and colocalizes with Nrf2 in human fetal eyes. Extracellular Sema6A leads to dose-dependent suppression of the migratory phenotype of endothelial cells through activation of Notch signaling. Lentiviral-mediated delivery of Sema6A small hairpin RNA (shRNA) abrogates the defective retinal revascularization in Nrf2-deficient mice. Importantly, pharmacologic Nrf2 activation promotes reparative angiogenesis and suppresses pathologic neovascularization. Our findings reveal a unique function of Nrf2 in reprogramming ischemic tissue toward neurovascular repair via Sema6A regulation, providing a potential therapeutic strategy for ischemic retinal and CNS diseases.

Purpose To determine if oral losartan treatment decreases the retinal ganglion cell (RGC) death caused by experimental intraocular pressure (IOP) elevation in mice. Methods We produced IOP increase in CD1 mice and performed unilateral optic nerve crush. Mice received oral losartan, spironolactone, enalapril, or no drug to test effects of inhibiting angiotensin receptors. IOP was monitored by Tonolab, and blood pressure was monitored by tail cuff device. RGC loss was measured in masked axon counts and RGC bodies by β-tubulin labeling. Scleral changes that could modulate RGC injury were measured including axial length, scleral thickness, and retinal layer thicknesses, pressure-strain behavior in inflation testing, and study of angiotensin receptors and pathways by reverse transcription polymerase chain reaction, Western blot, and immunohistochemistry. Results Losartan treatment prevented significant RGC loss (median loss = 2.5%, p = 0.13), while median loss with water, spironolactone, and enalapril treatments were 26%, 28% and 43%; p < 0.0001). The lower RGC loss with losartan was significantly less than the loss with spironolactone or enalapril (regression model p = 0.001; drug treatment group term p = 0.01). Both losartan and enalapril significantly lowered blood pressure (p< 0.001), but losartan was protective, while enalapril led to worse than water-treated RGC loss. RGC loss after crush injury was unaffected by losartan treatment (difference from control p = 0.9). Survival of RGC in cell culture was not prolonged by sartan treatment. Axonal transport blockade after 3 day IOP elevations was less in losartan-treated than in control glaucoma eyes (p = 0.007). Losartan inhibited effects of glaucoma, including reduction in extracellular signal-related kinase activity and modification of glaucoma-related changes in scleral thickness and creep under controlled IOP. Conclusions The neuroprotective effect of losartan in mouse glaucoma is associated with adaptive changes in the sclera expressed at the optic nerve head.

The androgen receptor not only mediates prostate development but also serves as a key regulator of primary prostatic cancer growth. Although initially responsive to selective androgen receptor modulators (SARMs), which cause recruitment of the nuclear receptor–corepressor (N-CoR) complex, resistance invariably occurs, perhaps in response to inflammatory signals. Here we report that dismissal of nuclear receptor–corepressor complexes by specific signals or androgen receptor overexpression results in recruitment of many of the cohorts of coactivator complexes that permits SARMs and natural ligands to function as agonists. SARM-bound androgen receptors appear to exhibit failure to recruit specific components of the coactivators generally bound by liganded nuclear receptors, including cAMP response element-binding protein (CBP)/p300 or coactivator-associated arginine methyltransferase 1 (CARM1) to the SARM-bound androgen receptor, although still causing transcriptional activation of androgen receptor target genes. SARM-bound androgen receptors use distinct LXXLL (L, leucine; X, any amino acid) helices in the p160 nuclear receptor interaction domains that may impose selective allosteric effects, providing a component of the molecular basis of differential responses to different classes of ligands by androgen receptor.

Inhibitors of the kinase mammalian target of rapamycin (mTOR) have shown sporadic activity in cancer trials, leading to confusion about the appropriate clinical setting for their use. Here we show that loss of the Von Hippel-Lindau tumor suppressor gene (VHL) sensitizes kidney cancer cells to the mTOR inhibitor CCI-779 in vitro and in mouse models. Growth arrest caused by CCI-779 correlates with a block in translation of mRNA encoding hypoxia-inducible factor (HIF1A), and is rescued by expression of a VHL-resistant HIF1A cDNA lacking the 5' untranslated region. VHL-deficient tumors show increased uptake of the positron emission tomography (PET) tracer fluorodeoxyglucose (FDG) in an mTOR-dependent manner. Our findings provide preclinical rationale for prospective, biomarker-driven clinical studies of mTOR inhibitors in kidney cancer and suggest that FDG-PET scans may have use as a pharmacodynamic marker in this setting.

In rats, feeding can be triggered experimentally using many approaches. Included among these are (1) food deprivation and (2) acute microinjection of the neurotransmitterl-glutamate (Glu) or its receptor agonist NMDA into the lateral hypothalamic area (LHA). Under both paradigms, the NMDA receptor (NMDA-R) within the LHA appears critically involved in transferring signals encoded by Glu to stimulate feeding. However, the intracellular mechanisms underlying this signal transfer are unknown. Because protein-tyrosine kinases (PTKs) participate in NMDA-R signaling mechanisms, we determined PTK involvement in LHA mechanisms underlying both types of feeding stimulation through food intake and biochemical measurements. LHA injections of PTK inhibitors significantly suppressed feeding elicited by LHA NMDA injection (up to 69%) but only mildly suppressed deprivation feeding (24%), suggesting that PTKs may be less critical for signals underlying this feeding behavior. Conversely, food deprivation but not NMDA injection produced marked increases in apparent activity for Src PTKs and in the expression of Pyk2, an Src-activating PTK. When considered together, the behavioral and biochemical results demonstrate that, although it is easier to suppress NMDA-elicited feeding by PTK inhibitors, food deprivation readily drives PTK activityin vivo. The latter result may reflect greater PTK recruitment by neurotransmitter receptors, distinct from the NMDA-R, that are activated during deprivation-elicited but not NMDA-elicited feeding. These results also demonstrate how the use of only one feeding stimulation paradigm may fail to reveal the true contributions of signaling molecules to pathways underlying feeding behaviorin vivo.

Using microarray-based profiling of isogenic prostate cancer xenograft models, we found that a modest increase in androgen receptor mRNA was the only change consistently associated with the development of resistance to antiandrogen therapy. This increase in androgen receptor mRNA and protein was both necessary and sufficient to convert prostate cancer growth from a hormone-sensitive to a hormone-refractory stage, and was dependent on a functional ligand-binding domain. Androgen receptor antagonists showed agonistic activity in cells with increased androgen receptor levels; this antagonist-agonist conversion was associated with alterations in the recruitment of coactivators and corepressors to the promoters of androgen receptor target genes. Increased levels of androgen receptor confer resistance to antiandrogens by amplifying signal output from low levels of residual ligand, and by altering the normal response to antagonists. These findings provide insight toward the design of new antiandrogens.

Retinal ischemia plays a critical role in multiple vision-threatening diseases and leads to death of retinal neurons, particularly ganglion cells. Oxidative stress plays an important role in this ganglion cell loss. Nrf2 (NF-E2-related factor 2) is a major regulator of the antioxidant response, and its role in the retina is increasingly appreciated. We investigated the potential retinal neuroprotective function of Nrf2 after ischemia-reperfusion (I/R) injury. In an experimental model of retinal I/R, Nrf2 knockout mice exhibited much greater loss of neuronal cells in the ganglion cell layer than wild-type mice. Primary retinal ganglion cells isolated from Nrf2 knockout mice exhibited decreased cell viability compared to wild-type retinal ganglion cells, demonstrating the cell-intrinsic protective role of Nrf2. The retinal neuronal cell line 661W exhibited reduced cell viability following siRNA-mediated knockdown of Nrf2 under conditions of oxidative stress, and this was associated with exacerbation of increase in reactive oxygen species. The synthetic triterpenoid CDDO-Im (2-Cyano-3,12-dioxooleana-1,9-dien-28-imidazolide), a potent Nrf2 activator, inhibited reactive oxygen species increase in cultured 661W under oxidative stress conditions and increased neuronal cell survival after I/R injury in wild-type, but not Nrf2 knockout mice. Our findings indicate that Nrf2 exhibits a retinal neuroprotective function in I/R and suggest that pharmacologic activation of Nrf2 could be a therapeutic strategy. Oxidative stress is thought to be an important mediator of retinal ganglion cell death in ischemia-reperfusion injury. We found that the transcription factor NF-E2-related factor 2 (Nrf2), a major regulator of oxidative stress, is an important endogenous neuroprotective molecule in retinal ganglion cells in ischemia-reperfusion, exerting a cell-autonomous protective effect. The triterpenoid 2-Cyano-3,12-dioxooleana-1,9-dien-28-imidazolide (CDDO-Im) reduces neurodegeneration following ischemia-reperfusion in an Nrf2-dependent fashion. This suggests that Nrf2-activating drugs including triterpenoids could be a therapeutic strategy for retinal neuroprotection.

Dual leucine zipper kinase (DLK) is a serine/threonine protein kinase that is a member of the mixed lineage kinase subfamily. Mixed lineage kinases are upstream MAP3Ks that activate the JNK pathway. DLK is primarily responsible for activating JNK and mediating the apoptotic stress response in various cell types, specifically neurons. Inhibition and knockdown of DLK has been demonstrated to have neuroprotective effects in cellular and animal models of Alzheimer’s disease, glaucoma, Parkinson’s disease and other neurodegenerative conditions. Several series of ATP-binding site inhibitors have been identified through profiling efforts providing launch points for future medicinal chemistry programs.

Vision loss from ischemic retinopathies commonly results from the accumulation of fluid in the inner retina [macular edema (ME)]. Although the precise events that lead to the development of ME remain under debate, growing evidence supports a role for an ischemia-induced hyperpermeability state regulated, in part, by VEGF. Monthly treatment with anti-VEGF therapies is effective for the treatment of ME but results in a major improvement in vision in a minority of patients, underscoring the need to identify additional therapeutic targets. Using the oxygen-induced retinopathy mouse model for ischemic retinopathy, we provide evidence showing that hypoxic Müller cells promote vascular permeability by stabilizing hypoxia-inducible factor-1α (HIF-1α) and secreting angiogenic cytokines. Blocking HIF-1α translation with digoxin inhibits the promotion of endothelial cell permeability in vitro and retinal edema in vivo. Interestingly, Müller cells require HIF--but not VEGF--to promote vascular permeability, suggesting that other HIF-dependent factors may contribute to the development of ME. Using gene expression analysis, we identify angiopoietin-like 4 (ANGPTL4) as a cytokine up-regulated by HIF-1 in hypoxic Müller cells in vitro and the ischemic inner retina in vivo. ANGPTL4 is critical and sufficient to promote vessel permeability by hypoxic Müller cells. Immunohistochemical analysis of retinal tissue from patients with diabetic eye disease shows that HIF-1α and ANGPTL4 localize to ischemic Müller cells. Our results suggest that ANGPTL4 may play an important role in promoting vessel permeability in ischemic retinopathies and could be an important target for the treatment of ME.

Glaucoma, a major cause of blindness worldwide, is a neurodegenerative optic neuropathy in which vision loss is caused by loss of retinal ganglion cells (RGCs). To better define the pathways mediating RGC death and identify targets for the development of neuroprotective drugs, we developed a high-throughput RNA interference screen with primary RGCs and used it to screen the full mouse kinome. The screen identified dual leucine zipper kinase (DLK) as a key neuroprotective target in RGCs. In cultured RGCs, DLK signaling is both necessary and sufficient for cell death. DLK undergoes robust posttranscriptional up-regulation in response to axonal injury in vitro and in vivo. Using a conditional knockout approach, we confirmed that DLK is required for RGC JNK activation and cell death in a rodent model of optic neuropathy. In addition, tozasertib, a small molecule protein kinase inhibitor with activity against DLK, protects RGCs from cell death in rodent glaucoma and traumatic optic neuropathy models. Together, our results establish a previously undescribed drug/drug target combination in glaucoma, identify an early marker of RGC injury, and provide a starting point for the development of more specific neuroprotective DLK inhibitors for the treatment of glaucoma, nonglaucomatous forms of optic neuropathy, and perhaps other CNS neurodegenerations.

A Second Act for Antiandrogens Men with advanced prostate cancer are often treated with antiandrogens; drugs that inhibit the activity of male hormones, such as testosterone, that help drive tumor growth. Many of these drugs act by functionally disrupting the androgen receptor (AR), a transcriptional regulator of cell proliferation, but tumors eventually become resistant to the drugs by expressing higher levels of the AR. Tran et al. (p. 787 , published online 9 April) have developed a “second-generation” antiandrogen, a thiohydantoin called MDV3100, which binds the AR with high affinity. MDV3100 retains its anticancer activity in cell culture and in mouse models even when AR levels are elevated. The drug appears to act both by inhibiting translocation of the AR into the nucleus and by reducing its transcriptional activity. MDV3100 is being tested in patients with advanced prostate cancer, the first group of which have shown a decline in blood levels of a marker of cancer growth, prostate-specific antigen.

One of the earliest steps in the development of the atherosclerotic lesion is the accumulation of monocyte/macrophages within the vessel wall. Oxidized lipids present in minimally modified-low density lipoproteins (MM-LDL) contribute to this process by activating endothelial cells to express monocyte-specific adhesion molecules and chemoattractant factors. A major focus of our group has been the isolation and characterization of the biologically active oxidized lipids in MM-LDL. We have previously characterized three oxidized phospholipids present in MM-LDL, atherosclerotic lesions of fat fed rabbits, and autoxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (Ox-PAPC) that induced human aortic endothelial cells to adhere human monocytes in vitro. We have used sequential normal and reverse phase-high performance liquid chromatography to isolate various isomers of an oxidized phospholipid from autoxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine. The fatty acid in the sn-2 position of this biologically active isomer and its dehydration product was released by phospholipase A(2) and characterized. Hydrogenation with platinum(IV) oxide/hydrogen suggested a cyclic moiety, and reduction with sodium borohydride suggested two reducible oxygen-containing groups in the molecule. The fragmentation pattern produced by electrospray ionization-collision induced dissociation-tandem mass spectrometry was consistent with a molecule resembling an E-ring prostaglandin with an epoxide at the 5,6 position. The structure of this lipid was confirmed by proton nuclear magnetic resonance spectroscopy analysis of the free fatty acid isolated from the dehydration product of m/z 828.5. Based on these studies, we arrived at the structure of the biologically active oxidized phospholipids as 1-palmitoyl-2-(5, 6-epoxyisoprostane E(2))-sn-glycero-3-phosphocholine. The identification of this molecule adds epoxyisoprostanes to the growing list of biologically active isoprostanes.