Sustained neurotrophic factor cotreatment enhances donor and host retinal ganglion cell survival in mice.

Retinal ganglion cells (RGCs) lack regenerative capacity in mammals, and their degeneration in glaucoma leads to irreversible blindness. Traditional RGC transplantation has been limited by poor survival rates of transplanted cells in the hostile microenvironment of a diseased retina. Our research identifies brain-derived neurotrophic factor (BDNF) and glial-derived neurotrophic factor (GDNF) as key elements in retinal development and RGC survival through in silico analysis of the single-cell transcriptome of developing human retinas. Although these factors are abundant during development, they diminish in adulthood. Here, we demonstrate that a slow-release formulation of BDNF and GDNF enhances RGC differentiation and survival in vitro and improves RGC transplantation outcomes in mouse models. This co-treatment increased survival and coverage of donor RGCs within the retina and enhanced neurite extension toward the optic nerve head. Lastly, this co-treatment showed neuroprotective effects on host RGCs, preserving retinal function in a model of optic neuropathy. Altogether, our findings suggest that manipulating the retinal microenvironment with slow-release neurotrophic factors holds promise in regenerative medicine for treating glaucoma and other optic neuropathies. This approach not only improves donor cell survival and integration but also provides a neuroprotective benefit to host cells, indicating a significant advancement for glaucoma therapies.

The Dynamic SecYEG Translocon.

The spatial and temporal coordination of protein transport is an essential cornerstone of the bacterial adaptation to different environmental conditions. By adjusting the protein composition of extra-cytosolic compartments, like the inner and outer membranes or the periplasmic space, protein transport mechanisms help shaping protein homeostasis in response to various metabolic cues. The universally conserved SecYEG translocon acts at the center of bacterial protein transport and mediates the translocation of newly synthesized proteins into and across the cytoplasmic membrane. The ability of the SecYEG translocon to transport an enormous variety of different substrates is in part determined by its ability to interact with multiple targeting factors, chaperones and accessory proteins. These interactions are crucial for the assisted passage of newly synthesized proteins from the cytosol into the different bacterial compartments. In this review, we summarize the current knowledge about SecYEG-mediated protein transport, primarily in the model organism Escherichia coli, and describe the dynamic interaction of the SecYEG translocon with its multiple partner proteins. We furthermore highlight how protein transport is regulated and explore recent developments in using the SecYEG translocon as an antimicrobial target.

Transplantation of miPSC/mESC-derived retinal ganglion cells into healthy and glaucomatous retinas.

Optic neuropathies, including glaucoma, are a group of neurodegenerative diseases, characterized by the progressive loss of retinal ganglion cells (RGCs), leading to irreversible vision loss. While previous studies demonstrated the potential to replace RGCs with primary neurons from developing mouse retinas, their use is limited clinically. We demonstrate successful transplantation of mouse induced pluripotent stem cell (miPSC)/mouse embryonic stem cell (mESC)-derived RGCs into healthy and glaucomatous mouse retinas, at a success rate exceeding 65% and a donor cell survival window of up to 12 months. Transplanted Thy1-GFP+ RGCs were able to polarize within the host retina and formed axonal processes that followed host axons along the retinal surface and entered the optic nerve head. RNA sequencing of donor RGCs re-isolated from host retinas at 24 h and 1 week post-transplantation showed upregulation of cellular pathways mediating axonal outgrowth, extension, and guidance. Additionally, we provide evidence of subtype-specific diversity within miPSC-derived RGCs prior to transplantation.

Eeyarestatin 24 impairs SecYEG-dependent protein trafficking and inhibits growth of clinically relevant pathogens.

Eeyarestatin 1 (ES1) is an inhibitor of endoplasmic reticulum (ER) associated protein degradation, Sec61-dependent Ca2+ homeostasis and protein translocation into the ER. Recently, evidence was presented showing that a smaller analog of ES1, ES24, targets the Sec61-translocon, and captures it in an open conformation that is translocation-incompetent. We now show that ES24 impairs protein secretion and membrane protein insertion in Escherichia coli via the homologous SecYEG-translocon. Transcriptomic analysis suggested that ES24 has a complex mode of action, probably involving multiple targets. Interestingly, ES24 shows antibacterial activity toward clinically relevant strains. Furthermore, the antibacterial activity of ES24 is equivalent to or better than that of nitrofurantoin, a known antibiotic that, although structurally similar to ES24, does not interfere with SecYEG-dependent protein trafficking. Like nitrofurantoin, we find that ES24 requires activation by the NfsA and NfsB nitroreductases, suggesting that the formation of highly reactive nitroso intermediates is essential for target inactivation in vivo.

Multifarious Biologic Loaded Liposomes that Stimulate the Mammalian Target of Rapamycin Signaling Pathway Show Retina Neuroprotection after Retina Damage.

A common event in optic neuropathies is the loss of axons and death of retinal ganglion cells (RGCs) resulting in irreversible blindness. Mammalian target of rapamycin (mTOR) signaling pathway agonists have been shown to foster axon regeneration and RGC survival in animal models of optic nerve damage. However, many challenges remain in developing therapies that exploit cell growth and tissue remodeling including (i) activating/inhibiting cell pathways synergistically, (ii) avoiding tumorigenesis, and (iii) ensuring appropriate physiological tissue function. These challenges are further exacerbated by the need to overcome ocular physiological barriers and clearance mechanisms. Here we present liposomes loaded with multiple mTOR pathway stimulating biologics designed to enhance neuroprotection after retina damage. Liposomes were loaded with ciliary neurotrophic factor, insulin-like growth factor 1, a lipopeptide N-fragment osteopontin mimic, and lipopeptide phosphatase tension homologue inhibitors for either the ATP domain or the c-terminal tail. In a mouse model of N-methyl-d-aspartic acid induced RGC death, a single intravitreal administration of liposomes reduced both RGC death and loss of retina electrophysiological function. Furthermore, combining liposomes with transplantation of induced pluripotent stem cell derived RGCs led to an improved electrophysiological outcome in mice. The results presented here show that liposomes carrying multiple signaling pathway modulators can facilitate neuroprotection and transplant electrophysiological outcome.

Regenerative medicine in the retina: from stem cells to cell replacement therapy.

Following the fast pace of the growing field of stem cell research, retinal cell replacement is finally emerging as a feasible mean to be explored for clinical application. Although neuroprotective treatments are able to slow the progression of retinal degeneration caused by diseases such as age-related macular degeneration and glaucoma, they are insufficient to fully halt disease progression and unable to recover previously lost vision. Comprehensive, technological and intellectual advances over the past years, including the in vitro differentiation of retinal cells at manufacturing scale from embryonic stem (ES) cell and induced pluripotent stem (iPS) cell cultures, progress within the area of retinal disease modeling, and the first clinical trials have started to shape the way towards addressing this treatment gap and translating retinal cell replacement to the clinic. Here, summarize the most recent advances within retinal cell replacement from both a scientific and clinical perspective, and discuss the remaining challenges towards the delivery of the first retinal cell products.

Müller glia provide essential tensile strength to the developing retina.

To investigate the cellular basis of tissue integrity in a vertebrate central nervous system (CNS) tissue, we eliminated Müller glial cells (MG) from the zebrafish retina. For well over a century, glial cells have been ascribed a mechanical role in the support of neural tissues, yet this idea has not been specifically tested in vivo. We report here that retinas devoid of MG rip apart, a defect known as retinoschisis. Using atomic force microscopy, we show that retinas without MG have decreased resistance to tensile stress and are softer than controls. Laser ablation of MG processes showed that these cells are under tension in the tissue. Thus, we propose that MG act like springs that hold the neural retina together, finally confirming an active mechanical role of glial cells in the CNS.

Cathepsin K deficiency in mice induces structural and metabolic changes in the central nervous system that are associated with learning and memory deficits.

BACKGROUND: Cathepsin K is a cysteine peptidase known for its importance in osteoclast-mediated bone resorption. Inhibitors of cathepsin K are in clinical trials for treatment of osteoporosis. However, side effects of first generation inhibitors included altered levels of related cathepsins in peripheral organs and in the central nervous system (CNS). Cathepsin K has been recently detected in brain parenchyma and it has been linked to neurobehavioral disorders such as schizophrenia. Thus, the study of the functions that cathepsin K fulfils in the brain becomes highly relevant. RESULTS: Cathepsin K messenger RNA was detectable in all brain regions of wild type (WT) mice. At the protein level, cathepsin K was detected by immunofluorescence microscopy in vesicles of neuronal and non-neuronal cells throughout the mouse brain. The hippocampus of WT mice exhibited the highest levels of cathepsin K activity in fluorogenic assays, while the cortex, striatum, and cerebellum revealed significantly lower enzymatic activities. At the molecular level, the proteolytic network of cysteine cathepsins was disrupted in the brain of cathepsin K-deficient (Ctsk⁻/⁻) animals. Specifically, cathepsin B and L protein and activity levels were altered, whereas cathepsin D remained largely unaffected. Cystatin C, an endogenous inhibitor of cysteine cathepsins, was elevated in the striatum and hippocampus, pointing to regional differences in the tissue response to Ctsk ablation. Decreased levels of astrocytic glial fibrillary acidic protein, fewer and less ramified profiles of astrocyte processes, differentially altered levels of oligodendrocytic cyclic nucleotide phosphodiesterase, as well as alterations in the patterning of neuronal cell layers were observed in the hippocampus of Ctsk⁻/⁻ mice. A number of molecular and cellular changes were detected in other brain regions, including the cortex, striatum/mesencephalon, and cerebellum. Moreover, an overall induction of the dopaminergic system was found in Ctsk⁻/⁻ animals which exhibited reduced anxiety levels as well as short- and long-term memory impairments in behavioral assessments. CONCLUSION: We conclude that deletion of the Ctsk gene can lead to deregulation of related proteases, resulting in a wide range of molecular and cellular changes in the CNS with severe consequences for tissue homeostasis. We propose that cathepsin K activity has an important impact on the development and maintenance of the CNS in mice.