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Abstract Mammalian central nervous system (CNS) axons cannot spontaneously regenerate after injury, creating an unmet need to identify molecular regulators to promote axon regeneration and reduce the lasting impact of CNS injuries. While tubulin polymerization promoting protein family member 3 (Tppp3) is known to promote axon outgrowth in amphibians, its role in mammalian axon regeneration remains unknown. Here we investigated Tppp3 in retinal ganglion cells (RGCs) neuroprotection and axonal regeneration using an optic nerve crush (ONC) model in the rodent. Single-cell RNA sequencing identified the expression of Tppp3 in RGCs of mice, macaques, and humans. Tppp3 overexpression enhanced neurite outgrowth in mouse primary RGCs in vitro, promoted axon regeneration, and improved RGC survival after ONC. Bulk RNA sequencing indicated that Tppp3 overexpression upregulates axon regeneration genes such as Bmp4 and neuroinflammatory pathways. Our findings advance regenerative medicine by developing a new therapeutic strategy for RGC neuroprotection and axon regeneration.

Summary: Retinal ganglion cell (RGC) differentiation is tightly controlled by extrinsic and intrinsic factors. Growth and differentiation factor 15 (GDF-15) promotes RGC differentiation, opposite to GDF-11 which inhibits RGC differentiation, both in the mouse retina and in human stem cells. To deepen our understanding of how these two closely related molecules confer opposing effects on retinal development, here we assess the transcriptional profiles of mouse retinal progenitors exposed to exogenous GDF-11 or -15. We find a dichotomous effect of GDF-15 on RGC differentiation, decreasing RGCs expressing residual pro-proliferative genes and increasing RGCs expressing non-proliferative genes, suggestive of greater RGC maturation. Furthermore, GDF-11 promoted the differentiation of photoreceptors and amacrine cells. These data enhance our understanding of the mechanisms underlying the differentiation of RGCs and photoreceptors from retinal progenitors and suggest new approaches to the optimization of protocols for the differentiation of these cell types.

Abstract Retinal ganglion cell (RGC) death in glaucoma and other optic neuropathies results in irreversible vision loss due to the mammalian central nervous system’s limited regenerative capacity. RGC repopulation is a promising therapeutic approach to reverse vision loss from optic neuropathies if the newly introduced neurons can reestablish functional retinal and thalamic circuits. In theory, RGCs might be repopulated through the transplantation of stem cell-derived neurons or via the induction of endogenous transdifferentiation. The RGC Repopulation, Stem Cell Transplantation, and Optic Nerve Regeneration (RReSTORe) Consortium was established to address the challenges associated with the therapeutic repair of the visual pathway in optic neuropathy. In 2022, the RReSTORe Consortium initiated ongoing international collaborative discussions to advance the RGC repopulation field and has identified five critical areas of focus: (1) RGC development and differentiation, (2) Transplantation methods and models, (3) RGC survival, maturation, and host interactions, (4) Inner retinal wiring, and (5) Eye-to-brain connectivity. Here, we discuss the most pertinent questions and challenges that exist on the path to clinical translation and suggest experimental directions to propel this work going forward. Using these five subtopic discussion groups (SDGs) as a framework, we suggest multidisciplinary approaches to restore the diseased visual pathway by leveraging groundbreaking insights from developmental neuroscience, stem cell biology, molecular biology, optical imaging, animal models of optic neuropathy, immunology & immunotolerance, neuropathology & neuroprotection, materials science & biomedical engineering, and regenerative neuroscience. While significant hurdles remain, the RReSTORe Consortium’s efforts provide a comprehensive roadmap for advancing the RGC repopulation field and hold potential for transformative progress in restoring vision in patients suffering from optic neuropathies.

Objectives. Primary cilia are conserved organelles found in polarized mammalian cells that regulate neuronal growth, migration, and differentiation. Proper cilia formation is essential during eye development. Our previous reports found that both amacrine and retinal ganglion cells (RGCs) contain primary cilia in primate and rodent retinas. However, whether primary cilia are present in the inner retina of human retinal organoids remains unknown. The purpose of this study is to characterize the primary cilia distribution in human embryonic stem cell (hESC-derived retinal organoid development. Materials and Methods. Retinal organoids were differentiated from a hESC line, harvested at various developmental timepoints (day 44-day 266), and immunostained with antibodies for primary cilia, including Arl13b (for the axoneme), AC3, and Centrin3 (for the basal body). AP2α, Prox1, GAD67, Calretinin, GFAP, PKCα, and Chx10 antibodies as well as Brn3b-promoted tdTomato expression were used to visualize retinal cell types. Results. A group of ciliated cells were present in the inner aspects of retinal organoids from day 44 to day 266 in culture. Ciliated Chx10-positive retinal progenitor cells, GFAP-positive astrocytes, and PKCα-positive rod-bipolar cells were detected later during development (day 176 to day 266). Ciliation persisted during all stages of retinal developmental in AP2α-positive amacrine cells, but it was decreased in Brn3b-positive retinal ganglion cells (RGCs) at later time points. Additionally, AC3-positive astrocytes significantly decreased during the later stages of organoid formation. Conclusions. Amacrine cells in retinal organoids retain cilia throughout development, whereas RGC ciliation gradually and progressively decreases with organoid maturation.

This paper addresses the problem of resilient average consensus in the presence of Byzantine agents in multi-agent networks. An event-triggered secure acceptance and broadcasting algorithm is proposed in which full knowledge of the network and high computational capabilities of each regular node are not required. The computational expense and communication times are also reduced for the event-triggered mechanism. We analyze the conditions for such a fully distributed algorithm to succeed in the f-local adversarial model. A new definition called an f-propagation graph, which is extended from r-robustness, turns out to be more accurate in describing the required topology conditions. Based on the proposed algorithm and topology conditions, we provide another algorithm to detect the adversarial nodes according to their abnormal behavior. When the network topology is an f-propagation graph, regular nodes that are equipped with the proposed algorithms update state values synchronously and eventually converge asymptotically to resilient average consensus. Simulation results are provided to verify the effectiveness of our proposed algorithms and the network topology conditions.

Based on analyzing the characteristics of transport layer security (TLS) protocol,a distributed automation malicious traffic detecting system based on machine learning was designed.The characteristics of encrypted malware traffic from TLS data,observable metadata and contextual flow data was extracted.Support vector machine,random forest and extreme gradient boosting were used to compare the performance of the mainstream malicious encryption traffic identification which realized the efficient detection of malicious encryption traffic,and verified the validity of the detection system of malicious encryption traffic.

Abstract Background BAM15 is a novel mitochondrial protonophore uncoupler capable of protecting mammals from acute renal ischemic-reperfusion injury and cold-induced microtubule damage. The purpose of our study was to investigate the effect of BAM15 on apoptosis during 5-day transportation of human-induced pluripotent stem (hiPS)-differentiated retinal tissue. Methods Retinal tissues of 30 days and 60 days were transported with or without BAM15 for 5 days in the laboratory or by real express. Immunofluorescence staining of apoptosis marker cleaved caspase3, proliferation marker Ki67, and neural axon marker NEFL was performed. And expression of apoptotic-related factors p53, NFkappaB, and TNF-a was detected by real-time PCR. Also, location of ganglion cells, photoreceptor cells, amacrine cells, and precursors of neuronal cell types in retinal tissue was stained by immunofluorescence after transportation. Furthermore, cell viability was assessed by CCK8 assay. Results Results showed transportation remarkably intensified expression of apoptotic factor cleaved caspase3, p53, NFkappaB, and TNF-a, which could be reduced by supplement of BAM15. In addition, neurons were severely injured after transportation, with axons manifesting disrupted and tortuous by staining NEFL. And the addition of BAM15 in transportation was able to protect neuronal structure and increase cell viability without affecting subtypes cells location of retinal tissue. Conclusions BAM15 might be used as a protective reagent on apoptosis during transporting retinal tissues, holding great potential in research and clinical applications.

This study was conducted to determine the dynamic Islet1 and Brn3 (POU4F) expression pattern in the human fetal retina and human-induced pluripotent stem cell- (hiPSC-) derived retinal organoid. Human fetal eyes from 8 to 27 fetal weeks (Fwks), human adult retina, hiPSC-derived retinal organoid from 7 to 31 differentiation weeks (Dwks), and rhesus adult retina were collected for cyrosectioning. Immunofluorescence analysis showed that Islet1 was expressed in retinal ganglion cells in the fetal retina, human adult retina, and retinal organoids. Unexpectedly, after Fwk 20, Brn3 expression gradually decreased in the fetal retina. In the midstage of development, Islet1 was detected in bipolar and developing horizontal cells. As the photoreceptor developed, the Islet1-positive cone precursors gradually became Islet1-negative/S-opsin-positive cones. This study highlights the distinguishing characteristics of Islet1 dynamic expression in human fetal retina development and proposes more concerns which should be taken regarding Brn3 as a cell-identifying marker in mature primate retina.

We investigated the efficacy of the immunosuppressants rapamycin (RAP) and dexamethasone (DEX) in improving the survival of retinal organoids after epiretinal transplantation. We first compared the immunosuppressive abilities of DEX and RAP in activated microglia in an in vitro setting. Following this, we used immunofluorescence, real-time polymerase chain reaction, and flow cytometry to investigate the effects of DEX and RAP on cells in the retinal organoids. Retinal organoids were then seeded onto poly(lactic-co-glycolic) acid (PLGA) scaffolds and implanted into rhesus monkey eyes (including a healthy individual and three monkeys with chronic ocular hypertension (OHT) induction) and subjected to different post-operative immunosuppressant treatments; 8 weeks after the experiment, histological examinations were carried out to assess the success of the different treatments. Our in vitro experiments indicated that both DEX and RAP treatments were equally effective in suppressing microglial activity. Although both immunosuppressants altered the morphologies of cells in the retinal organoids and caused a slight decrease in the differentiation of cells into retinal ganglion cells, the organoid cells retained their capacity to grow and differentiate into retinal tissues. Our in vivo experiments indicate that the retinal organoid can survive and differentiate into retinal tissues in a healthy rhesus monkey eye without immunosuppressive treatment. However, the survival and differentiation of these organoids in OHT eyes was successful only with the DEX treatment. RAP treatment was ineffective in preventing immunological rejection, and the retinal organoid failed to survive until the end of 8 weeks. DEX is likely a promising immunosuppressant to enhance the survival of epiretinal implants.

Background. To develop an effective surgical procedure for cellular scaffold epiretinal implantation in rhesus, facilitating subsequent epiretinal stem cell transplantation. Methods. Retinal progenitors were seeded onto a poly(lactic-co-glycolic) acid (PLGA) scaffold. First, the cellular scaffolds were delivered by 18G catheter or retinal forceps into rabbit epiretinal space (n=50). Then, the cell survival rate was evaluated by Cell Counting Kit-8 (CCK-8). Second, three methods of scaffold fixation, including adhesion after gas-liquid exchange (n=1), tamponade by hydrogel (n=1), and fixation by retinal tacks (n=4), were performed in rhesus monkeys. After one month, fundus photography and SD-OCT were performed to assess the outcomes, and histological examination was performed to evaluate proliferation. Results. The cell survival rate was significantly higher in the catheter group. Follow-up examination showed that retinal tack fixation was the only method to maintain the scaffolds attached to host retina for at least 3 weeks, which is the minimal time required for cell integration. Histological staining demonstrated slight glial fibrillary acidic protein (GFAP) accumulation in the retinal tack insertion area. Conclusions. The established surgical procedure offers a new insight into research of epiretinal cell replacement therapy in rhesus eyes. The successful delivery and long-term fixation provide a prerequisite for cell migration and integration.