Yuliya Naumchuk

Antioxidant Drug Therapy Approaches for Neuroprotection in Chronic Diseases of the Retina

The molecular pathways contributing to visual signal transduction in the retina generate a high energy demand that has functional and structural consequences such as vascularization and high metabolic rates contributing to oxidative stress. Multiple signaling cascades are involved to actively regulate the redox state of the retina. Age-related processes increase the oxidative load, resulting in chronically elevated levels of oxidative stress and reactive oxygen species, which in the retina ultimately result in pathologies such as glaucoma or age-related macular degeneration, as well as the neuropathic complications of diabetes in the eye. Specifically, oxidative stress results in deleterious changes to the retina through dysregulation of its intracellular physiology, ultimately leading to neurodegenerative and potentially also vascular dysfunction. Herein we will review the evidence for oxidative stress-induced contributions to each of the three major ocular pathologies, glaucoma, age-related macular degeneration, and diabetic retinopathy. The premise for neuroprotective strategies for these ocular disorders will be discussed in the context of recent clinical and preclinical research pursuing novel therapy development approaches.

Presenilins regulate the cellular activity of ryanodine receptors differentially through isotype-specific N-terminal cysteines.

Presenilins (PS), endoplasmic reticulum (ER) transmembrane proteins, form the catalytic core of γ-secretase, an amyloid precursor protein processing enzyme. Mutations in PS lead to Alzheimers disease (AD) by altering γ-secretase activity to generate pathologic amyloid beta and amyloid plaques in the brain. Here, we identified a novel mechanism where binding of a soluble, cytosolic N-terminal domain fragment (NTF) of PS to intracellular Ca(2+) release channels, ryanodine receptors (RyR), controls Ca(2+) release from the ER. While PS1NTF decreased total RyR-mediated Ca(2+) release, PS2NTF had no effect at physiological Ca(2+) concentrations. This differential function and isotype-specificity is due to four cysteines absent in PS1NTF, present, however, in PS2NTF. Site-directed mutagenesis targeting these cysteines converted PS1NTF to PS2NTF function and vice versa, indicating differential RyR binding. This novel mechanism of intracellular Ca(2+) regulation through the PS-RyR interaction represents a novel target for AD drug development and the treatment of other neurodegenerative disorders that critically depend on RyR and PS signaling.

Differential subcellular Ca2+ signaling in a highly specialized subpopulation of astrocytes.

Recent evidence suggests that astrocytes do not serve a mere buffering function, but exhibit complex signaling pathways, disturbance of which contributes significantly to the pathophysiology of CNS diseases. Little is known regarding the intracellular signaling pathways in the specialized optic nerve head astrocytes (ONHAs), the major glia cell type in non-myelinated optic nerve head. Here we show the differential subcellular expression of intracellular Ca(2+) channels in ONHAs. Expression of type 1 and type 3 inositol-1-4-5,-trisphosphate receptors (IP3Rs) in the endoplasmic reticulum and type 2 IP3Rs in the nuclear envelope causes differential Ca(2+) release from intracellular stores in nuclear vs. cytosolic compartments. Our study identifies differential distribution and activity of Ca(2+) channels as molecular substrate and mechanism by which astrocytes independently regulate Ca(2+) transients in both cytoplasm and nucleoplasm, thereby controlling genomic and non-genomic cellular signaling, respectively. This provides excellent targets for therapeutics restoring pathological disturbances of intracellular Ca(2+) signaling present in glaucoma and other neurodegenerative disorders with astrocyte involvement.

Differential up-regulation of Vesl-1/Homer 1 protein isoforms associated with decline in visual performance in a preclinical glaucoma model

Glaucoma is a multifactorial progressive ocular pathology, clinically presenting with damage to the retina and optic nerve, ultimately leading to blindness. Retinal ganglion cell loss in glaucoma ultimately results in vision loss. Vesl/Homer proteins are scaffolding proteins that are critical for maintaining synaptic integrity by clustering, organizing and functionally regulating synaptic proteins. Current anti-glaucoma therapies target IOP as the sole modifiable clinical parameters. Long-term pharmacotherapy and surgical treatment do not prevent gradual visual field loss as the disease progresses, highlighting the need for new complementary, alternative and comprehensive treatment approaches. Vesl/Homer expression was measured in the retinae of DBA/2J mice, a preclinical genetic glaucoma model with spontaneous mutations resulting in a phenotype reminiscent of chronic human pigmentary glaucoma. Vesl/Homer proteins were differentially expressed in the aged, glaucomatous DBA/2J retina, both at the transcriptional and translational level. Immunoreactivity for the long Vesl-1L/Homer 1c isoform, but not of the immediate early gene product Vesl-1S/Homer 1a was increased in the synaptic layers of the retina. This increased protein level of Vesl-1L/Homer 1c was correlated with phenotypes of increased disease severity and a decrease in visual performance. The increased expression of Vesl-1L/Homer 1c in the glaucomatous retina likely results in increased intracellular Ca(2+) release through enhancement of synaptic coupling. The ensuing Ca(2+) toxicity may thus activate neurodegenerative pathways and lead to the progressive loss of synaptic function in glaucoma. Our data suggest that higher levels of Vesl-1L/Homer 1c generate a more severe disease phenotype and may represent a viable target for therapy development.

nampt/PBeF/visfatin serum levels: a new biomarker for retinal blood vessel occlusions

The main objective of the study was to quantify serum levels of nicotinamide phosphoribosyltransferase (Nampt/pre-B-Cell colony-enhancing factor 1/visfatin) in subjects with a history of retinal vascular occlusions (RVOs), disease conditions characterized by pronounced ischemia, and metabolic energy deficits. A case–control study of 18 subjects with a history of RVO as well as six healthy volunteers is presented. Serum Nampt levels were quantified using a commercially available enzyme-linked immunosorbent assay kit. Serum Nampt levels were 79% lower in patients with a history of RVO compared with that in healthy volunteers (P<0.05). There was no statistically significant difference among the types of RVOs, specifically branch retinal vein occlusions (n=7), central retinal vein occlusions (n=5), hemiretinal vein occlusions (n=3), and central retinal artery occlusions (n=3; P=0.69). Further studies are needed to establish the temporal kinetics of Nampt expression and to determine whether Nampt may represent a novel biomarker to identify at-risk populations, or whether it is a druggable target with the potential to ameliorate the long-term complications associated with the condition, ie, macular edema, macular ischemia, neovascularization, and permanent loss of vision.

Quantification of Lactate Dehydrogenase for Cell Viability Testing Using Cell Lines and Primary Cultured Astrocytes

Drug discovery heavily relies on cell viability studies to assess the potential toxicity of drug candidates. L‐Lactate dehydrogenase (LDH) is a cytoplasmic enzyme that catalyzes the concomitant interconversions of pyruvate to L‐lactate and NADH to NAD+ during glycolysis, and the reverse reactions during the Cori cycle. In response to cellular damage, induced by endogenous cellular mechanisms or as a result of exogenously applied insults, LDH is released from the cytoplasm into the extracellular environment. Its stability in cell culture medium makes it a well‐suited correlate for the presence of damage and toxicity in tissues and cells. We herein present protocols for a reproducible and validated LDH assay optimized for several cell types. In contrast to commercially available LDH assays, often associated with proprietary formulations and high cost, our protocols provide ample opportunities for experiment‐specific optimization with low variability and cost.

Mobile Technology in Tele-education

Mobile communication has revolutionized the way of business and the practice of medicine alike. The smartphone has become an indispensable tool for healthcare providers by offering resources and tools helping to improve medical care, diagnostics, patient education, and personal productivity. In this chapter, we (1) summarize the history of smartphones and their impact on the field of tele-medicine; (2) showcase some of the software applications (apps) available on the smartphone for ophthalmologists, vision researchers, and patients; and (3) present the emerging use of the smartphone as research and diagnostic tools. We conclude that smartphones can significantly increase productivity in the clinical and laboratory setting and have great potential to become portable research and diagnostic tools.