Claudio Punzo

Stimulation of the insulin/mTOR pathway delays cone death in a mouse model of retinitis pigmentosa

Retinitis pigmentosa is an incurable retinal disease that leads to blindness. One puzzling aspect concerns the progression of the disease. Although most mutations that cause retinitis pigmentosa are in rod photoreceptor–specific genes, cone photoreceptors also die as a result of such mutations. To understand the mechanism of non-autonomous cone death, we analyzed four mouse models harboring mutations in rod-specific genes. We found changes in the insulin/mammalian target of rapamycin pathway that coincided with the activation of autophagy during the period of cone death. We increased or decreased the insulin level and measured the survival of cones in one of the models. Mice that were treated systemically with insulin had prolonged cone survival, whereas depletion of endogenous insulin had the opposite effect. These data suggest that the non-autonomous cone death in retinitis pigmentosa could, at least in part, be a result of the starvation of cones.

Loss of Daylight Vision in Retinal Degeneration: Are Oxidative Stress and Metabolic Dysregulation to Blame?

Retinitis pigmentosa is characterized by loss of night vision, followed by complete blindness. Over 40 genetic loci for retinitis pigmentosa have been identified in humans, primarily affecting photoreceptor structure and function. The availability of excellent animal models allows for a mechanistic characterization of the disease. Metabolic dysregulation and oxidative stress have been found to correlate with the loss of vision, particularly in cones, the type of photoreceptors that mediate daylight and color vision. The evidence that these problems actually cause loss of vision and potential therapeutic approaches targeting them are discussed.

Comparison of hybridization-based and sequencing-based gene expression technologies on biological replicates

BackgroundHigh-throughput systems for gene expression profiling have been developed and have matured rapidly through the past decade. Broadly, these can be divided into two categories: hybridization-based and sequencing-based approaches. With data from different technologies being accumulated, concerns and challenges are raised about the level of agreement across technologies. As part of an ongoing large-scale cross-platform data comparison framework, we report here a comparison based on identical samples between one-dye DNA microarray platforms and MPSS (Massively Parallel Signature Sequencing).ResultsThe DNA microarray platforms generally provided highly correlated data, while moderate correlations between microarrays and MPSS were obtained. Disagreements between the two types of technologies can be attributed to limitations inherent to both technologies. The variation found between pooled biological replicates underlines the importance of exercising caution in identification of differential expression, especially for the purposes of biomarker discovery.ConclusionBased on different principles, hybridization-based and sequencing-based technologies should be considered complementary to each other, rather than competitive alternatives for measuring gene expression, and currently, both are important tools for transcriptome profiling.

Transcription factor Olig2 defines subpopulations of retinal progenitor cells biased toward specific cell fates

Previous lineage analyses have shown that retinal progenitor cells (RPCs) are multipotent throughout development, and expression-profiling studies have shown a great deal of molecular heterogeneity among RPCs. To determine if the molecular heterogeneity predicts that an RPC will produce particular types of progeny, clonal lineage analysis was used to investigate the progeny of a subset of RPCs, those that express the basic helix–loop–helix transcription factor, Olig2. The embryonic Olig2+ RPCs underwent terminal divisions, producing small clones with primarily two of the five cell types being made by the pool of RPCs at that time. The later, postnatal Olig2+ RPCs also made terminal divisions, which were biased toward production of rod photoreceptors and amacrine cell interneurons. These data indicate that the multipotent progenitor pool is made up of distinctive types of RPCs, which have biases toward producing subsets of retinal neurons in a terminal division, with the types of neurons produced varying over time. This strategy is similar to that of the developing Drosophila melanogaster ventral nerve cord, with the Olig2+ cells behaving as ganglion mother cells.

Functional divergence between eyeless and twin of eyeless in Drosophila melanogaster

Pax6 genes encode transcription factors with two DNA-binding domains that are highly conserved during evolution. In Drosophila, two Pax6 genes function in a pathway in which twin of eyeless (toy) directly regulates eyeless (ey), which is necessary for initiating the eye developmental pathway. To investigate the gene duplication of Pax6 that occurred in holometabolous insects like Drosophila and silkworm, we used different truncated forms of toy and small eyes (sey), and tested their capacity to induce ectopic eye development in an ey-independent manner. Even though the Paired domains of TOY and SEY have DNA-binding properties that differ from those of the Paired domain of EY, they all are capable of inducing ectopic eye development in an ey mutant background. We also show that one of the main functional differences between toy and ey lies in the C-terminal region of their protein products, implying differences in their transactivation potential. Furthermore, we show that only the homeodomain (HD) of EY is able to downregulate the expression of Distal-less (Dll), a feature that is required during endogenous eye development. These results suggest distinct functions of the two DNA-binding domains of TOY and EY, and significant evolutionary divergence between the two Drosophila Pax6 genes.

Cross-regulatory protein–protein interactions between Hox and Pax transcription factors

Homeotic Hox selector genes encode highly conserved transcriptional regulators involved in the differentiation of multicellular organisms. Ectopic expression of the Antennapedia (ANTP) homeodomain protein in Drosophila imaginal discs induces distinct phenotypes, including an antenna-to-leg transformation and eye reduction. We have proposed that the eye loss phenotype is a consequence of a negative posttranslational control mechanism because of direct protein–protein interactions between ANTP and Eyeless (EY). In the present work, we analyzed the effect of various ANTP homeodomain mutations for their interaction with EY and for head development. Contrasting with the eye loss phenotype, we provide evidence that the antenna-to-leg transformation involves ANTP DNA-binding activity. In a complementary genetic screen performed in yeast, we isolated mutations located in the N terminus of the ANTP homeodomain that inhibit direct interactions with EY without abolishing DNA binding in vitro and in vivo. In a bimolecular fluorescence complementation assay, we detected the ANTP–EY interaction in vivo, these interactions occurring through the paired domain and/or the homeodomain of EY. These results demonstrate that the homeodomain supports multiple molecular regulatory functions in addition to protein–DNA and protein–RNA interactions; it is also involved in protein–protein interactions.

Activated mTORC1 promotes long-term cone survival in retinitis pigmentosa mice

Retinitis pigmentosa (RP) is an inherited photoreceptor degenerative disorder that results in blindness. The disease is often caused by mutations in genes that are specific to rod photoreceptors; however, blindness results from the secondary loss of cones by a still unknown mechanism. Here, we demonstrated that the mammalian target of rapamycin complex 1 (mTORC1) is required to slow the progression of cone death during disease and that constitutive activation of mTORC1 in cones is sufficient to maintain cone function and promote long-term cone survival. Activation of mTORC1 in cones enhanced glucose uptake, retention, and utilization, leading to increased levels of the key metabolite NADPH. Moreover, cone death was delayed in the absence of the NADPH-sensitive cell death protease caspase 2, supporting the contribution of reduced NADPH in promoting cone death. Constitutive activation of mTORC1 preserved cones in 2 mouse models of RP, suggesting that the secondary loss of cones is caused mainly by metabolic deficits and is independent of a specific rod-associated mutation. Together, the results of this study address a longstanding question in the field and suggest that activating mTORC1 in cones has therapeutic potential to prolong vision in RP.

Store-operated channels regulate intracellular calcium in mammalian rods

•  Light closes cyclic nucleotide‐gated and voltage operated calcium channels in vertebrate rod photoreceptors, resulting in a decrease in the intracellular calcium concentration ([Ca2+]i). A protracted decrease in [Ca2+]i experienced under saturating illuminations is toxic for these cells. •  Eukaryotic cells express voltage‐independent plasma membrane ion channels that protect against pathological [Ca2+]i decreases and can be activated by depletion of intracellular calcium stores. An invertebrate homologue of canonical transient receptor potential (TRPC) channels that have been implicated in store‐operated calcium entry (SOCE) in vertebrates is expressed in photoreceptors. •  We show that mouse rods express a potent SOCE mechanism that gates cation entry which subsequently modulates activation of L‐type calcium channels. Furthermore, we show what the majority of the retinal Trpc1 signal is localized to rod photoreceptors. •  We found, using knockout animal models, that neither TRPC1 nor TRPC3 channels contribute to SOCE in mouse rod perikarya, or regulate light‐evoked responses in the outer segment and the synaptic terminal, suggesting that the channels are receptor operated. •  We conclude that mammalian rods express two new calcium signalling mechanisms associated with SOCE and TRPC1 signalling which modulate calcium homeostasis and may protect against prolonged [Ca2+]i decreases in saturating light.

Ultrasound-Guided In Utero Injections Allow Studies of the Development and Function of the Eye

Ultrasound‐guided in utero injections into the brain of murine embryos has been shown to facilitate gene delivery. We investigated whether these methods would allow gene transfer into ocular structures. Gene transfer using retroviral vectors or electroporation was found to be quite effective. We determined the window of time, as well as compared several strains of mice, that yield a high degree of survival and successful gene transfer. Several retroviral constructs were tested for expression and coexpresssion of two genes in retinal cell types. In addition, a retroviral vector was engineered to give cone photoreceptor‐enriched expression, and a retroviral vector was demonstrated to provide RNAi‐mediated loss‐of‐function. These methods enable access to early ocular structures and provide a more rapid method of assessment of gene and promoter function than possible using genetically engineered mice. Developmental Dynamics 237:1034–1042, 2008.

In Vivo Selection Yields AAV-B1 Capsid for Central Nervous System and Muscle Gene Therapy

Adeno-associated viral (AAV) vectors have shown promise as a platform for gene therapy of neurological disorders. Achieving global gene delivery to the central nervous system (CNS) is key for development of effective therapies for many of these diseases. Here we report the isolation of a novel CNS tropic AAV capsid, AAV-B1, after a single round of in vivo selection from an AAV capsid library. Systemic injection of AAV-B1 vector in adult mice and cat resulted in widespread gene transfer throughout the CNS with transduction of multiple neuronal subpopulations. In addition, AAV-B1 transduces muscle, β-cells, pulmonary alveoli, and retinal vasculature at high efficiency. This vector is more efficient than AAV9 for gene delivery to mouse brain, spinal cord, muscle, pancreas, and lung. Together with reduced sensitivity to neutralization by antibodies in pooled human sera, the broad transduction profile of AAV-B1 represents an important improvement over AAV9 for CNS gene therapy.