Retinitis pigmentosa: rapid neurodegeneration is governed by slow cell death mechanisms

For most neurodegenerative diseases the precise duration of an individual cell's death is unknown, which is an obstacle when counteractive measures are being considered. To address this, we used the rd1 mouse model for retinal neurodegeneration, characterized by phosphodiesterase-6 (PDE6) dysfunction and photoreceptor death triggered by high cyclic guanosinemono-phosphate (cGMP) levels. Using cellular data on cGMP accumulation, cell death, and survival, we created mathematical models to simulate the temporal development of the degeneration. We validated model predictions using organotypic retinal explant cultures derived from wild-type animals and exposed to the selective PDE6 inhibitor zaprinast. Together, photoreceptor data and modeling for the first time delineated three major cell death phases in a complex neuronal tissue: (1) initiation, taking up to 36 h, (2) execution, lasting another 40 h, and finally (3) clearance, lasting about 7 h. Surprisingly, photoreceptor neurodegeneration was noticeably slower than necrosis or apoptosis, suggesting a different mechanism of death for these neurons. Cell Death and Disease (2013) 4, e488; doi: 10.1038/cddis.2013.12; published online 7 February 201

Efficient Delivery of Hydrophilic Small Molecules to Retinal Cell Lines Using Gel Core-Containing Solid Lipid Nanoparticles

In this study, we developed a novel solid lipid nanoparticle (SLN) formulation for drug delivery of small hydrophilic cargos to the retina. The new formulation, based on a gel core and composite shell, allowed up to two-fold increase in the encapsulation efficiency. The type of hydrophobic polyester used in the composite shell mixture affected the particle surface charge, colloidal stability, and cell internalization profile. We validated SLNs as a drug delivery system by performing the encapsulation of a hydrophilic neuroprotective cyclic guanosine monophosphate analog, previously demonstrated to hold retinoprotective properties, and the best formulation resulted in particles with a size of ±250 nm, anionic charge > −20 mV, and an encapsulation efficiency of ±60%, criteria that are suitable for retinal delivery. In vitro studies using the ARPE-19 and 661W retinal cell lines revealed the relatively low toxicity of SLNs, even when a high particle concentration was used. More importantly, SLN could be taken up by the cells and the release of the hydrophilic cargo in the cytoplasm was visually demonstrated. These findings suggest that the newly developed SLN with a gel core and composite polymer/lipid shell holds all the characteristics suitable for the drug delivery of small hydrophilic active molecules into retinal cells

Contribution of the cyclic nucleotide gated channel subunit, CNG-3, to olfactory plasticity in Caenorhabditis elegans.

In Caenorhabditis elegans, the AWC neurons are thought to deploy a cGMP signaling cascade in the detection of and response to AWC sensed odors. Prolonged exposure to an AWC sensed odor in the absence of food leads to reversible decreases in the animal's attraction to that odor. This adaptation exhibits two stages referred to as short-term and long-term adaptation. Previously, the protein kinase G (PKG), EGL-4/PKG-1, was shown necessary for both stages of adaptation and phosphorylation of its target, the beta-type cyclic nucleotide gated (CNG) channel subunit, TAX-2, was implicated in the short term stage. Here we uncover a novel role for the CNG channel subunit, CNG-3, in short term adaptation. We demonstrate that CNG-3 is required in the AWC for adaptation to short (thirty minute) exposures of odor, and contains a candidate PKG phosphorylation site required to tune odor sensitivity. We also provide in vivo data suggesting that CNG-3 forms a complex with both TAX-2 and TAX-4 CNG channel subunits in AWC. Finally, we examine the physiology of different CNG channel subunit combinations

Light-Driven Calcium Signals in Mouse Cone Photoreceptors

Calcium mediates various neuronal functions. The complexity of neuronal Ca2+ signaling is well exemplified by retinal cone photoreceptors, which, with their distinct compartmentalization, offer unique possibilities for studying the diversity of Ca2+ functions in a single cell. Measuring subcellular Ca2+ signals in cones under physiological conditions is not only fundamental for understanding cone function, it also bears important insights into pathophysiological processes governing retinal neurodegeneration. However, due to the proximity of light-sensitive outer segments to other cellular compartments, optical measurements of light-evoked Ca2+ responses in cones are challenging. We addressed this problem by generating a transgenic mouse (HR2.1:TN-XL) in which both short-and middle-wavelength-sensitive cones selectively express the genetically encoded ratiometric Ca2+ biosensor TN-XL. We show that HR2.1:TN-XL allows recording of light-evoked Ca2+ responses using two-photon imaging in individual cone photoreceptor terminals and to probe phototransduction and its diverse regulatory mechanisms with pharmacology at subcellular resolution. To further test this system, we asked whether the classical, nitric oxide (NO)-soluble guanylyl-cyclase (sGC)-cGMP pathway could modulate Ca2 in cone terminals. Surprisingly, NO reduced Ca2+ resting levels in mouse cones, without evidence for direct sGC involvement. In conclusion, HR2.1:TN-XL mice offer unprecedented opportunities to elucidate light-driven Ca2+ dynamics and their (dys) regulation in cone photoreceptors