Adam N. Baker

PEG-PLA microparticles for encapsulation and delivery of Tat-EGFP to retinal cells.

The efficient and controlled delivery of genes and proteins to retinal cells remains a challenge. In this study, we evaluated polyethylene glycol-polylactic acid (PEG-PLA) microparticles for encapsulation and delivery of a Transactivator of transcription-enhanced green fluorescent protein fusion (Tat-EGFP) to retinal cells. Our main objective was to develop a microparticle system that delivers Tat-EGFP with an initial rapid release (within 24 h) followed by a sustained release. We prepared four different formulations of Tat-EGFP encapsulated PEG-PLA particles to investigate the effects of protein and polymer concentrations on particle morphology and protein release, using scanning electron microscopy (SEM) and fluorometry techniques. The optimum formulation was selected based on higher protein release, and smaller particle size. The optimum formulation was then tested in vitro for cell biocompatibility and protein internalization, and in vivo for cellular toxicity following sub-retinal injections into rat eyes. The results suggest that PEG-PLA microparticles can deliver proteins in cell culture allowing protein internalization in as little as 1 h. In vivo, protein was shown to localize within the photoreceptor layer of the retina, and persist for at least 9 weeks with no observed toxicity.

Effects on XIAP Retinal Detachment-Induced Photoreceptor Apoptosis

PURPOSE To evaluate the ability of X-linked inhibitor of apoptosis (XIAP) gene therapy to provide neuroprotection to cells of the outer nuclear layer (ONL) of the retina after retinal detachment. METHODS Subretinal injections of a recombinant adenoassociated virus (rAAV) encoding either XIAP or green fluorescent protein (GFP; injection control) were performed in the left eye of Brown Norway rats. Two weeks later, retinal detachments were created at the site of viral injection by delivering sodium hyaluronate into the subretinal space. Retinal tissue was harvested at 24 hours after retinal detachment and was analyzed for caspase 3 and 9 activity. Histologic analysis was conducted on samples taken at 3 days and 2 months after detachment to confirm the presence of XIAP or GFP expression and to assess levels of apoptosis and changes in retinal thickness. RESULTS Caspase assays performed 24 hours after detachment confirmed an expected increase in caspase 3 and 9 activity in the detached regions of GFP-treated retinas, whereas XIAP-treated detached retinas behaved comparably to attached controls. TUNEL analysis of 3-day tissue samples showed fewer apoptotic cells in XIAP-treated detachments than in GFP-treated detachments. At 2 months after the detachment, histology and immunohistochemistry confirmed the preservation of the ONL at sites of XIAP overexpression, whereas the GFP-treated detached retinas had significantly deteriorated. CONCLUSIONS The results suggest that XIAP confers structural neuroprotection of photoreceptors for at least 2 months after retinal detachment.

Smn deficiency causes neuritogenesis and neurogenesis defects in the retinal neurons of a mouse model of spinal muscular atrophy

The eye is an excellent model for the study of neuronal development and pathogenesis of central nervous system disorders because of its relative ease of accessibility and the well‐characterized cellular makeup. We have used this model to study spinal muscular atrophy (SMA), an autosomal recessive neuromuscular disease caused by deletions or mutations in the survival of motor neuron 1 gene (SMN1). We have investigated the expression pattern of mouse Smn mRNA and protein in the neural retina and the optic nerve of wild type mice. Smn protein is present in retinal ganglion cells and amacrine cells within the neural retina as well as in glial cells in the optic nerve. Histopathological analysis in phenotype stage SMA mice revealed that Smn deficiency is associated with a reduction in ganglion cell axon and glial cell number in the optic nerve, as well as compromised cellular processes and altered organization of neurofilaments in the neural retina. Whole mount preparation and retinal neuron primary culture provided further evidence of abnormal synaptogenesis and neurofilament accumulation in the neurites of Smn‐deficient retinal neurons. A subset of amacrine cells is absent, in a cell‐autonomous fashion, in the retina of SMA mice. Finally, the retinas of SMA mice have altered electroretinograms. Altogether, our study has demonstrated defects in axodendritic outgrowth and cellular composition in Smn‐depleted retinal neurons, indicating a role for Smn in neuritogenesis and neurogenesis, and providing us with an insight into pathogenesis of SMA.

Establishment of a cone photoreceptor transplantation platform based on a novel cone-GFP reporter mouse line.

We report successful retinal cone enrichment and transplantation using a novel cone-GFP reporter mouse line. Using the putative cone photoreceptor-enriched transcript Coiled-Coil Domain Containing 136 (Ccdc136) GFP-trapped allele, we monitored developmental reporter expression, facilitated the enrichment of cones, and evaluated transplanted GFP-labeled cones in wildtype and retinal degeneration mutant retinas. GFP reporter and endogenous Ccdc136 transcripts exhibit overlapping temporal and spatial expression patterns, both initiated in cone precursors of the embryonic retina and persisting to the adult stage in S and S/M opsin+ cones as well as rod bipolar cells. The trapped allele does not affect cone function or survival in the adult mutant retina. When comparing the integration of GFP+ embryonic cones and postnatal Nrl−/− ‘cods’ into retinas of adult wildtype and blind mice, both cell types integrated and exhibited a degree of morphological maturation that was dependent on donor age. These results demonstrate the amenability of the adult retina to cone transplantation using a novel transgenic resource that can advance therapeutic cone transplantation in models of age-related macular degeneration.

Chitosan microparticles for delivery of proteins to the retina.

Chitosan microparticles (CMPs) have previously been developed for topical applications to the eye, but their safety and efficacy in delivering proteins to the retina have not been adequately evaluated. This study examines the release kinetics of CMPs in vitro, and assesses their biocompatibility and cytotoxicity on retinal cells in vitro and in vivo. Two proteins were used in the encapsulation and release studies: BSA (bovine serum albumin) and tat-EGFP (enhanced green fluorescent protein fused to the transactivator of transcription peptide). Not surprisingly, the in vitro release kinetics were dependent on the protein encapsulated, with BSA showing higher release than tat-EGFP. CMPs containing encapsulated tat-EGFP were tested for cellular toxicity in photoreceptor-derived 661W cells. They showed no signs of in vitro cell toxicity at a low concentration (up to 1mgml(-1)), but at a higher concentration of 10mgml(-1) they were associated with cytotoxic effects. In vivo, CMPs injected into the subretinal space were found beneath the photoreceptor layer of the retina, and persisted for at least 8weeks. Similar to the in vitro studies, the lower concentration of CMPs was generally well tolerated, but the higher concentration resulted in cytotoxic effects and in reduced retinal function, as assessed by electroretinogram amplitudes. Overall, this study suggests that CMPs are effective long-term delivery agents to the retina, but the concentration of chitosan may affect cytotoxicity.

Erratum: Corrigendum: Establishment of a cone photoreceptor transplantation platform based on a novel cone-GFP reporter mouse line

Scientific Reports 6: Article number: 22867 ; published online: 11 March 2016; updated: 22 April 2016 The original version of this Article contained a typographical error in the spelling of the author Yves De Repentigny, which was incorrectly given as Yves De Repentingy. This has now been corrected in the PDF and HTML versions of the Article.

Retinal interneuron survival requires non-cell-autonomous Atrx activity

ATRX is a chromatin remodeling protein that is mutated in several intellectual disability disorders including alpha-thalassemia/mental retardation, X-linked (ATR-X) syndrome. We previously reported the prevalence of ophthalmological defects in ATR-X syndrome patients, and accordingly we find morphological and functional visual abnormalities in a mouse model harboring a mutation occurring in ATR-X patients. The visual system abnormalities observed in these mice parallels the Atrx-null retinal phenotype characterized by interneuron defects and selective loss of amacrine and horizontal cells. The mechanisms that underlie selective neuronal vulnerability and neurodegeneration in the central nervous system upon Atrx mutation or deletion are unknown. To interrogate the cellular specificity of Atrx for its retinal neuroprotective functions, we employed a combination of temporal and lineage-restricted conditional ablation strategies to generate five different conditional knockout mouse models, and subsequently identified a non-cell-autonomous requirement for Atrx in bipolar cells for inhibitory interneuron survival in the retina. Atrx-deficient retinal bipolar cells exhibit functional, structural and molecular alterations consistent with impairments in neuronal activity and connectivity. Gene expression changes in the Atrx-null retina indicate defective synaptic structure and neuronal circuitry, suggest excitotoxic mechanisms of neurodegeneration, and demonstrate that common targets of ATRX in the forebrain and retina may contribute to similar neuropathological processes underlying cognitive impairment and visual dysfunction in ATR-X syndrome.