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PurposeTo 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.MethodsThe 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.ResultsMechanical 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.ConclusionsThe 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.

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.

PrcisWe 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.PurposeTo evaluate clinician perceptions of a prototyped clinical decision support (CDS) tool that integrates visual field (VF) metric predictions from artificial intelligence (AI) models.MethodsTen 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 measuresMean 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.ResultsThe 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).ConclusionsA 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.

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.

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.

PurposePolymorphous 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.ObservationsA 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.ConclusionsThere 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.

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.

PurposeTo 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).MethodsFor 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.ResultsIncreased 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.ConclusionsHuman 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.

PurposeTo 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).MethodsFor 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.ResultsIncreased 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.ConclusionsHuman 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.

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.

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 1 day, 1 week, 6 weeks, 3 months, and 6 months postoperatively. Subconjunctival 5-fluorouracil injections were given postoperatively, as needed. The primary outcome was the proportion of patients who demonstrated IOP of

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.

BackgroundTraumatic 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.MethodsHere 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.ResultsOur 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.ConclusionThe 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.

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.

Author(s): George V Thomas [1]; Chris Tran [2]; Ingo K Mellinghoff [3, 4, 5]; Derek S Welsbie [3]; Emily Chan [1]; Barbara Fueger [4]; Johannes Czernin [4]; Charles L Sawyers [...]

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., Author(s): Charlie D Chen [1, 5, 8]; Derek S Welsbie [3, 5, 8]; Chris Tran [1, 4]; Sung Hee Baek [4, 6]; Randy Chen [1]; Robert Vessella [7]; Michael G [...]

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. [ABSTRACT FROM AUTHOR]