Inhibition of GCK-IV kinases dissociates cell death and axon regeneration in CNS neurons.

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.

Aqueous shunts with mitomycin C versus aqueous shunts alone for glaucoma.

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.

Role of SARM1 and DR6 in retinal ganglion cell axonal and somal degeneration following axonal injury.

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.

Nrf2 in ischemic neurons promotes retinal vascular regeneration through regulation of semaphorin 6A.

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.

Characterization of intravitreally delivered capsid mutant AAV2-Cre vector to induce tissue-specific mutations in murine retinal ganglion cells.

Targeted expression of Cre recombinase in murine retinal ganglion cells (RGCs) by viral vector is an effective strategy for creating tissue-specific gene knockouts for investigation of genetic contribution to RGC degeneration associated with optic neuropathies. Here we characterize dosage, efficacy and toxicity for sufficient intravitreal delivery of a capsid mutant Adeno-associated virus 2 (AAV2) vector encoding Cre recombinase. Wild type and Rosa26 (R26) LacZ mice were intravitreally injected with capsid mutant AAV2 viral vectors. Murine eyes were harvested at intervals ranging from 2 weeks to 15 weeks post-injection and were assayed for viral transduction, transgene expression and RGC survival. 10(9) vector genomes (vg) were sufficient for effective in vivo targeting of murine ganglion cell layer (GCL) retinal neurons. Transgene expression was observed as early as 2 weeks post-injection of viral vectors and persisted to 11 weeks. Early expression of Cre had no significant effect on RGC survival, while significant RGC loss was detected beginning 5 weeks post-injection. Early expression of viral Cre recombinase was robust, well-tolerated and predominantly found in GCL neurons suggesting this strategy can be effective in short-term RGC-specific mutation studies in experimental glaucoma models such as optic nerve crush and transection experiments. RGC degeneration with Cre expression for more than 4 weeks suggests that Cre toxicity is a limiting factor for targeted mutation strategies in RGCs.

Hypoxic retinal Muller cells promote vascular permeability by HIF-1-dependent up-regulation of angiopoietin-like 4.

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.

Functional genomic screening identifies dual leucine zipper kinase as a key mediator of retinal ganglion cell death.

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.

Neuroprotective role of Nrf2 for retinal ganglion cells in ischemia-reperfusion.

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.

Optineurin-facilitated axonal mitochondria delivery promotes neuroprotection and axon regeneration.

Optineurin (OPTN) mutations are linked to amyotrophic lateral sclerosis (ALS) and normal tension glaucoma (NTG), but a relevant animal model is lacking, and the molecular mechanisms underlying neurodegeneration are unknown. We found that OPTN C-terminus truncation (OPTN∆C) causes late-onset neurodegeneration of retinal ganglion cells (RGCs), optic nerve (ON), and spinal cord motor neurons, preceded by a striking decrease of axonal mitochondria. Surprisingly, we discover that OPTN directly interacts with both microtubules and the mitochondrial transport complex TRAK1/KIF5B, stabilizing them for proper anterograde axonal mitochondrial transport, in a C-terminus dependent manner. Encouragingly, overexpressing OPTN/TRAK1/KIF5B reverses not only OPTN truncation-induced, but also ocular hypertension-induced neurodegeneration, and promotes striking ON regeneration. Therefore, in addition to generating new animal models for NTG and ALS, our results establish OPTN as a novel facilitator of the microtubule-dependent mitochondrial transport necessary for adequate axonal mitochondria delivery, and its loss as the likely molecular mechanism of neurodegeneration.

Hypoxia-inducible factor determines sensitivity to inhibitors of mTOR in kidney cancer.

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.

Usability and Clinician Acceptance of a Deep Learning-Based Clinical Decision Support Tool for Predicting Glaucomatous Visual Field Progression.

PRCIS: We updated a clinical decision support tool integrating predicted visual field (VF) metrics from an artificial intelligence model and assessed clinician perceptions of the predicted VF metric in this usability study. PURPOSE: To evaluate clinician perceptions of a prototyped clinical decision support (CDS) tool that integrates visual field (VF) metric predictions from artificial intelligence (AI) models. METHODS: Ten ophthalmologists and optometrists from the University of California San Diego participated in 6 cases from 6 patients, consisting of 11 eyes, uploaded to a CDS tool ("GLANCE", designed to help clinicians "at a glance"). For each case, clinicians answered questions about management recommendations and attitudes towards GLANCE, particularly regarding the utility and trustworthiness of the AI-predicted VF metrics and willingness to decrease VF testing frequency. MAIN OUTCOMES AND MEASURES: Mean counts of management recommendations and mean Likert scale scores were calculated to assess overall management trends and attitudes towards the CDS tool for each case. In addition, system usability scale scores were calculated. RESULTS: The mean Likert scores for trust in and utility of the predicted VF metric and clinician willingness to decrease VF testing frequency were 3.27, 3.42, and 2.64, respectively (1=strongly disagree, 5=strongly agree). When stratified by glaucoma severity, all mean Likert scores decreased as severity increased. The system usability scale score across all responders was 66.1±16.0 (43rd percentile). CONCLUSIONS: A CDS tool can be designed to present AI model outputs in a useful, trustworthy manner that clinicians are generally willing to integrate into their clinical decision-making. Future work is needed to understand how to best develop explainable and trustworthy CDS tools integrating AI before clinical deployment.

A Deep Learning Approach to Improve Retinal Structural Predictions and Aid Glaucoma Neuroprotective Clinical Trial Design.

PURPOSE: 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. DESIGN: Cross-sectional study. PARTICIPANTS: 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. METHODS: 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. MAIN OUTCOME MEASURES: Mean absolute error (MAE) and squared Pearson correlation coefficient (r2) were used to evaluate model performance. RESULTS: The deep learning model was able to predict superior and inferior GCIPL thicknesses with a global r2 value of 0.90 and 0.86, r2 of mean of 0.90 and 0.86, and mean MAE of 3.72 µm and 4.2 µm, respectively. For superior and inferior RNFL thickness predictions, model performance was slightly lower, with a global r2 of 0.75 and 0.84, r2 of mean of 0.81 and 0.82, and MAE of 9.31 µm and 8.57 µm, respectively. There was only a modest decrease in model performance when predicting GCIPL and RNFL in more severe disease. Using individualized hemiretinal predictions to account for variability across patients, we estimate that a clinical trial can detect a difference equivalent to a 25% treatment effect over 24 months with an 11-fold reduction in the number of patients compared to a conventional trial. CONCLUSIONS: Our 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. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found after the references.

Mechanical Disruption of the Inner Limiting Membrane In Vivo Enhances Targeting to the Inner Retina.

Purpose: To evaluate the effects of mechanical disruption of the inner limiting membrane (ILM) on the ability to target interventions to the inner neurosensory retina in a rodent model. Our study used an animal model to gain insight into the normal physiology of the ILM and advances our understanding of the effects of mechanical ILM removal on the viral transduction of retinal ganglion cells and retinal ganglion cell transplantation. Methods: The ILM in the in vivo rat eye was disrupted using mechanical forces applied to the vitreoretinal interface. Immunohistology and electron microscopy were used to verify the removal of the ILM in retina flatmounts and sections. To assess the degree to which ILM disruption enhanced transvitreal access to the retina, in vivo studies involving intravitreal injections of adeno-associated virus (AAV) to transduce retinal ganglion cells (RGCs) and ex vivo studies involving co-culture of human stem cell-derived RGCs (hRGCs) on retinal explants were performed. RGC transduction efficiency and transplanted hRGC integration with retinal explants were evaluated by immunohistology of the retinas. Results: Mechanical disruption of the ILM in the rodent eye was sufficient to remove the ILM from targeted retinal areas while preserving the underlying retinal nerve fiber layer and RGCs. Removal of the ILM enhanced the transduction efficiency of intravitreally delivered AAV threefold (1380.0 ± 290.1 vs. 442.0 ± 249.3 cells/mm2; N = 6; P = 0.034). Removal of the ILM was also sufficient to promote integration of transplanted RGCs within the inner retina. Conclusions: The ILM is a barrier to transvitreally delivered agents including viral vectors and cells. Mechanical removal of the ILM is sufficient to enhance access to the inner retina, improve viral transduction efficiencies of RGCs, and enhance cellular integration of transplanted RGCs with the retina.

Osteopontin drives retinal ganglion cell resiliency in glaucomatous optic neuropathy.

Chronic neurodegeneration and acute injuries lead to neuron losses via diverse processes. We compared retinal ganglion cell (RGC) responses between chronic glaucomatous conditions and the acute injury model. Among major RGC subclasses, αRGCs and intrinsically photosensitive RGCs (ipRGCs) preferentially survive glaucomatous conditions, similar to findings in the retina subject to axotomy. Focusing on an αRGC intrinsic factor, Osteopontin (secreted phosphoprotein 1 [Spp1]), we found an ectopic neuronal expression of Osteopontin (Spp1) in other RGCs subject to glaucomatous conditions. This contrasted with the Spp1 downregulation subject to axotomy. αRGC-specific Spp1 elimination led to significant αRGC loss, diminishing their resiliency. Spp1 overexpression led to robust neuroprotection of susceptible RGC subclasses under glaucomatous conditions. In contrast, Spp1 overexpression did not significantly protect RGCs subject to axotomy. Additionally, SPP1 marked adult human RGC subsets with large somata and SPP1 expression in the aqueous humor correlated with glaucoma severity. Our study reveals Spp1's role in mediating neuronal resiliency in glaucoma.

Human retinal ganglion cell neurons generated by synchronous BMP inhibition and transcription factor mediated reprogramming.

In optic neuropathies, including glaucoma, retinal ganglion cells (RGCs) die. Cell transplantation and endogenous regeneration offer strategies for retinal repair, however, developmental programs required for this to succeed are incompletely understood. To address this, we explored cellular reprogramming with transcription factor (TF) regulators of RGC development which were integrated into human pluripotent stem cells (PSCs) as inducible gene cassettes. When the pioneer factor NEUROG2 was combined with RGC-expressed TFs (ATOH7, ISL1, and POU4F2) some conversion was observed and when pre-patterned by BMP inhibition, RGC-like induced neurons (RGC-iNs) were generated with high efficiency in just under a week. These exhibited transcriptional profiles that were reminiscent of RGCs and exhibited electrophysiological properties, including AMPA-mediated synaptic transmission. Additionally, we demonstrated that small molecule inhibitors of DLK/LZK and GCK-IV can block neuronal death in two pharmacological axon injury models. Combining developmental patterning with RGC-specific TFs thus provided valuable insight into strategies for cell replacement and neuroprotection.

Live, die, or regenerate? New insights from multi-omic analyses.

In this issue of Neuron, three studies establish new strategies to uncover mediators of retinal neuroprotection and optic nerve regeneration. Tian et al. (2022) carry out a multi-omics screen and identify transcriptional regulators of axon injury signaling leading to cell death; Jacobi et al. (2022) and Li et al. (2022) combine retrograde tracing and single-cell RNA-seq (scRNA-seq) to uncover molecular targets for axon regeneration.

Superior segmental optic nerve hypoplasia: A review.

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.

Polymorphous low-grade adenocarcinoma with cavernous sinus involvement presenting as third nerve palsy.

Purpose: Polymorphous low-grade adenocarcinoma is a tumor of the salivary glands that typically localizes within the oral cavity. We present a case of isolated third cranial nerve palsy as the initial presentation of polymorphous low-grade adenocarcinoma involving the left cavernous sinus in a patient status post glaucoma surgery. Observations: A 68-year-old woman status post glaucoma drainage device implantation in her left eye presented with an isolated left third nerve palsy ten weeks postoperatively. Differential diagnoses included microvascular ischemic neuropathy, postoperative ptosis, and compressive mass. MRI revealed a left cavernous sinus mass, and subsequent excisional biopsy revealed a diagnosis of polymorphous low-grade adenocarcinoma. Conclusions: There are few cases reporting polymorphous low-grade adenocarcinoma originating from and extending beyond the nasopharynx. This report emphasizes an unexpected neuro-ophthalmic manifestation of this salivary gland tumor.

Molecular determinants of resistance to antiandrogen therapy.

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.

Optic Nerve Engraftment of Neural Stem Cells.

Purpose: To evaluate the integrative potential of neural stem cells (NSCs) with the visual system and characterize effects on the survival and axonal regeneration of axotomized retinal ganglion cells (RGCs). Methods: For in vitro studies, primary, postnatal rat RGCs were directly cocultured with human NSCs or cultured in NSC-conditioned media before their survival and neurite outgrowth were assessed. For in vivo studies, human NSCs were transplanted into the transected rat optic nerve, and immunohistology of the retina and optic nerve was performed to evaluate RGC survival, RGC axon regeneration, and NSC integration with the injured visual system. Results: Increased neurite outgrowth was observed in RGCs directly cocultured with NSCs. NSC-conditioned media demonstrated a dose-dependent effect on RGC survival and neurite outgrowth in culture. NSCs grafted into the lesioned optic nerve modestly improved RGC survival following an optic nerve transection (593 ± 164 RGCs/mm2 vs. 199 ± 58 RGCs/mm2; P < 0.01). Additionally, RGC axonal regeneration following an optic nerve transection was modestly enhanced by NSCs transplanted at the lesion site (61.6 ± 8.5 axons vs. 40.3 ± 9.1 axons, P < 0.05). Transplanted NSCs also differentiated into neurons, received synaptic inputs from regenerating RGC axons, and extended axons along the transected optic nerve to incorporate with the visual system. Conclusions: Human NSCs promote the modest survival and axonal regeneration of axotomized RGCs that is partially mediated by diffusible NSC-derived factors. Additionally, NSCs integrate with the injured optic nerve and have the potential to form neuronal relays to restore retinofugal connections.