An endogenous PI3K interactome promoting astrocyte-mediated neuroprotection identifies a novel association with RNA-binding protein ZC3H14.

Astrocytes can support neuronal survival through a range of secreted signals that protect against neurotoxicity, oxidative stress, and apoptotic cascades. Thus, analyzing the effects of the astrocyte secretome may provide valuable insight into these neuroprotective mechanisms. Previously, we characterized a potent neuroprotective activity mediated by retinal astrocyte conditioned media (ACM) on retinal and cortical neurons in metabolic stress models. However, the molecular mechanism underlying this complex activity in neuronal cells has remained unclear. Here, a chemical genetics screen of kinase inhibitors revealed phosphoinositide 3-kinase (PI3K) as a central player transducing ACM-mediated neuroprotection. To identify additional proteins contributing to the protective cascade, endogenous PI3K was immunoprecipitated from neuronal cells exposed to ACM or control media, followed by MS/MS proteomic analyses. These data pointed toward a relatively small number of proteins that coimmunoprecipitated with PI3K, and surprisingly only five were regulated by the ACM signal. These hits included expected PI3K interactors, such as the platelet-derived growth factor receptor A (PDGFRA), as well as novel RNA-binding protein interactors ZC3H14 (zinc finger CCCH-type containing 14) and THOC1 (THO complex protein 1). In particular, ZC3H14 has recently emerged as an important RNA-binding protein with multiple roles in posttranscriptional regulation. In validation studies, we show that PI3K recruitment of ZC3H14 is necessary for PDGF-induced neuroprotection and that this interaction is present in primary retinal ganglion cells. Thus, we identified a novel non-cell autonomous neuroprotective signaling cascade mediated through PI3K that requires recruitment of ZC3H14 and may present a promising strategy to promote astrocyte-secreted prosurvival signals.

All roads lead to glaucoma: Induced retinal injury cascades contribute to a common neurodegenerative outcome.

Glaucoma describes a distinct optic neuropathy with complex etiology and a variety of associated risk factors, but with similar pathological endpoints. Risk factors such as age, increased intraocular pressure (IOP), low mean arterial pressure, and autoimmune disease, can all be associated with death of retinal ganglion cells (RGCs) and optic nerve head remodeling. Today, IOP management remains the standard of care, even though IOP elevation is not pathognomonic of glaucoma, and patients can continue to lose vision despite effective IOP control. A contemporary view of glaucoma as a complex, neurodegenerative disease has developed, along with the recognition of a need for new disease modifying retinal treatment strategies and improved outcomes. However, the distinction between risk factors triggering the disease process and retinal injury responses is not always clear. In this review, we attempt to distinguish between the various triggers, and their association with subsequent key RGC injury mechanisms. We propose that distinct glaucomatous risk factors result in similar retinal and optic nerve injury cascades, including oxidative and metabolic stress, glial reactivity, and altered inflammatory responses, which induce common molecular signals to induce RGC apoptosis. This organization forms a coherent disease framework and presents conserved targets for therapeutic intervention that are not limited to specific risk factors.

The AMPK-PGC-1α signaling axis regulates the astrocyte glutathione system to protect against oxidative and metabolic injury.

Neurons are highly sensitive to metabolic and oxidative injury, but endogenous astrocyte mechanisms have a critical capacity to provide protection from these stresses. We previously reported that the master regulator PGC-1α (peroxisome proliferator-activated receptor gamma coactivator-1α) is necessary for retinal astrocytes to mount effective injury responses, with particular regard to oxidative stress. Yet, this pathway has not been well studied in glia. PGC-1α is a transcriptional co-activator that is dysregulated in a variety of neurodegenerative diseases. It functions as a master regulator of cellular bioenergetics, with the ability to regulate tissue specific responses. A key inducer of PGC-1α signaling is adenosine monophosphate-activated kinase (AMPK). Thus, the AMPK-PGC-1α signaling axis coordinates metabolic and oxidative damage responses in the central nervous system (CNS). Here we report that AMPK selectively regulates expression of GCLM (glutamate cysteine ligase modulatory subunit) in astrocytes, but not neurons, through PGC-1α activation. Glutamate cysteine ligase (GCL) is the rate limiting enzyme in the biosynthesis of glutathione (GSH); a critical antioxidant and detoxifying peptide in the CNS. Through this mechanism we describe PGC-1α-dependent induction of GSH synthesis and antioxidant activity in astrocytes, and in the rodent retina in vivo. Furthermore, we demonstrate that therapeutic agonism of this pathway with the AMP mimetic, AICAR, rescues GSH levels in vivo, while reducing RGC death and astrocyte reactivity, following retinal ischemia/reperfusion injury. This mechanism presents a novel strategy for enhancing protective astrocyte antioxidant capacity in the CNS.

Preclinical development and ocular biodistribution of gemini-DNA nanoparticles after intravitreal and topical administration: towards non-invasive glaucoma gene therapy.

Gene therapy could offer improvement in the treatment of glaucoma compared to the current standard of lowering intraocular pressure. We have developed and characterized non-viral gemini surfactant-phospholipid nanoparticles (GL-NPs) for intravitreal and topical administration. Optimized GL-NPs (size range 150-180 nm) were biocompatible with rat retinal ganglion (RGC-5) cells with >95% viability by PrestoBlue™ assay. GL-NPs carrying Cy5-labeled plasmid DNA demonstrated distinct trafficking behavior and biodisposition within the eye in vivo after intravitreal or topical application with respect to pathways of movement and physicochemical stability. After intravitreal injection in mice, GL-NPs localized within the nerve fiber layer of the retina, whereas after topical application, GL-NPs were located in several anterior chamber tissues, including the limbus, iris and conjunctiva. GL-NPs were thermodynamically stable in the vitreous and tear fluid and were trafficked as single, non-aggregated particles after both types of administration. FROM THE CLINICAL EDITOR: In this paper, the development and characterization of non-viral gemini surfactant-phospholipid nanoparticles is reported with the goal of establishing a gene delivery system that addresses glaucoma in a non-invasive fashion. The authors found that after topical application, the concentration of these nanoparticles was higher in anterior chamber-related components of the eye, whereas intra-vitreal administration resulted in accumulation in the retinal nerve fibre layer.

Astrocyte-derived lipoxins A4 and B4 promote neuroprotection from acute and chronic injury.

Astrocytes perform critical non-cell autonomous roles following CNS injury that involve either neurotoxic or neuroprotective effects. Yet the nature of potential prosurvival cues has remained unclear. In the current study, we utilized the close interaction between astrocytes and retinal ganglion cells (RGCs) in the eye to characterize a secreted neuroprotective signal present in retinal astrocyte conditioned medium (ACM). Rather than a conventional peptide neurotrophic factor, we identified a prominent lipid component of the neuroprotective signal through metabolomics screening. The lipoxins LXA4 and LXB4 are small lipid mediators that act locally to dampen inflammation, but they have not been linked directly to neuronal actions. Here, we determined that LXA4 and LXB4 are synthesized in the inner retina, but their levels are reduced following injury. Injection of either lipoxin was sufficient for neuroprotection following acute injury, while inhibition of key lipoxin pathway components exacerbated injury-induced damage. Although LXA4 signaling has been extensively investigated, LXB4, the less studied lipoxin, emerged to be more potent in protection. Moreover, LXB4 neuroprotection was different from that of established LXA4 signaling, and therapeutic LXB4 treatment was efficacious in a chronic model of the common neurodegenerative disease glaucoma. Together, these results identify a potential paracrine mechanism that coordinates neuronal homeostasis and inflammation in the CNS.

DNA ministrings: highly safe and effective gene delivery vectors.

Conventional plasmid DNA vectors play a significant role in gene therapy, but they also have considerable limitations: they can elicit adverse immune responses because of bacterial sequences they contain for maintenance and amplification in prokaryotes, their bioavailability is compromised because of their large molecular size, and they may be genotoxic. We constructed an in vivo platform to produce ministring DNA-mini linear covalently closed DNA vectors-that are devoid of unwanted bacterial sequences and encode only the gene(s) of interest and necessary eukaryotic expression elements. Transfection of rapidly and slowly dividing human cells with ministring DNA coding for enhanced green fluorescent protein resulted in significantly improved transfection, bioavailability, and cytoplasmic kinetics compared with parental plasmid precursors and isogenic circular covalently closed DNA counterparts. Ministring DNA that integrated into the genome of human cells caused chromosomal disruption and apoptotic death of possibly oncogenic vector integrants; thus, they may be safer than plasmid and circular DNA vectors.

Challenges in neuroprotective nanomedicine development: progress towards noninvasive gene therapy of glaucoma.

Over the past decade the application of gene therapy of retinal diseases such as glaucoma has produced promising results. However, optic nerve regeneration and restoration of vision in patients with glaucoma is still far from reality. Neuroprotective approaches in the form of gene therapy may provide significant advantages, but are still limited by many factors both at the organ and cellular levels. In general, gene delivery systems for eye diseases range from simple eye drops and ointments to more advanced bio- and nanotechnology-based systems such as muco-adhesive systems, polymers, liposomes and ocular inserts. Most of these technologies were developed for front-of-the-eye ophthalmic therapies and are not applicable as back-of-the-eye delivery systems. Currently, only the invasive intravitreal injections are capable of successfully delivering genes to the retina. Here we review the challenges and possible strategies for the noninvasive gene therapy of glaucoma including the barriers in the eye and in neural cells, and present a cross-sectional view of gene delivery as it pertains to the prevention and treatment of glaucoma.

Is color patchy?

In many natural scenes, shadows and shading, which are primarily luminance-defined features, proliferate. Hence one might expect that the chromatic variations of natural scenes, which more faithfully represent the layout of object surfaces, will contain relatively fewer and larger uniform regions than the luminance variations, i.e., will be more "patchy." This idea was tested using images of natural scenes that were decomposed into chromatic and luminance layers modeled as the responses of the red-green, blue-yellow, and luminance channels of the human visual system. Patchiness was defined as the portion of pixels falling within a +/- threshold in the bandpass-filtered image, averaged across multiple filter scales. The red-green layers were found to be the most patchy, followed by the blue-yellow layers, with the luminance layers the least patchy. The correlation between image-layer patchiness and the slope of the Fourier amplitude spectrum was small and negative for all layers, the maximum value being for red-green (-0.48). We conclude that the chromatic layers of natural scenes contain more uniform areas than the luminance layers and that this is unpredicted by the slope of the Fourier amplitude spectrum.