Tiziana Cogliati

Update on the Kelch-like (KLHL) gene family

The Kelch-like (KLHL) gene family encodes a group of proteins that generally possess a BTB/POZ domain, a BACK domain, and five to six Kelch motifs. BTB domains facilitate protein binding and dimerization. The BACK domain has no known function yet is of functional importance since mutations in this domain are associated with disease. Kelch domains form a tertiary structure of β-propellers that have a role in extracellular functions, morphology, and binding to other proteins. Presently, 42 KLHL genes have been classified by the HUGO Gene Nomenclature Committee (HGNC), and they are found across multiple human chromosomes. The KLHL family is conserved throughout evolution. Phylogenetic analysis of KLHL family members suggests that it can be subdivided into three subgroups with KLHL11 as the oldest member and KLHL9 as the youngest. Several KLHL proteins bind to the E3 ligase cullin 3 and are known to be involved in ubiquitination. KLHL genes are responsible for several Mendelian diseases and have been associated with cancer. Further investigation of this family of proteins will likely provide valuable insights into basic biology and human disease.

Glutamate transporters in platelets: EAAT1 decrease in aging and in Alzheimer's disease

Platelets release glutamate upon activation and are an important clearance system of the amino acid from blood, through high-affinity glutamate uptake, similar to that described in brain synaptosomes. Since platelet glutamate uptake is decreased in neurodegenerative disorders, we performed a morphological and molecular characterization of platelet glutamate transporters. The three major brain glutamate transporters EAAT1, EAAT2 and EAAT3 are expressed in platelets, with similar molecular weight, although at lower density than brain. A Na(+)-dependent-high-affinity glutamate uptake was competitively inhibited by known inhibitors but not by dihydrokainic acid, suggesting platelet EAAT2 does not play a major role in glutamate uptake at physiological conditions. We observed decreased glutamate uptake V(max), without modification of transporter affinity, in aging, which could be linked to the selective decrease of EAAT1 expression and mRNA. Moreover, in AD patients we found a further EAAT1 reduction compared to age-matched controls, which could explain the decrease of platelet uptake previously described. Platelet glutamate transporters may be used as peripheral markers to investigate the role of glutamate in patients with neuropsychiatric disorders.

Developing Rods Transplanted into the Degenerating Retina of Crx-Knockout Mice Exhibit Neural Activity Similar to Native Photoreceptors†‡§

Replacement of dysfunctional or dying photoreceptors offers a promising approach for retinal neurodegenerative diseases, including age‐related macular degeneration and retinitis pigmentosa. Several studies have demonstrated the integration and differentiation of developing rod photoreceptors when transplanted in wild‐type or degenerating retina; however, the physiology and function of the donor cells are not adequately defined. Here, we describe the physiological properties of developing rod photoreceptors that are tagged with green fluorescent protein (GFP) driven by the promoter of rod differentiation factor, Nrl. GFP‐tagged developing rods show Ca2 + responses and rectifier outward currents that are smaller than those observed in fully developed photoreceptors, suggesting their immature developmental state. These immature rods also exhibit hyperpolarization‐activated current (Ih) induced by the activation of hyperpolarization‐activated cyclic nucleotide‐gated (HCN) channels. When transplanted into the subretinal space of wild‐type or retinal degeneration mice, GFP‐tagged developing rods can integrate into the photoreceptor outer nuclear layer in wild‐type mouse retina and exhibit Ca2 + responses and membrane current comparable to native rod photoreceptors. A proportion of grafted rods develop rhodopsin‐positive outer segment‐like structures within 2 weeks after transplantation into the retina of Crx‐knockout mice and produce rectifier outward current and Ih upon membrane depolarization and hyperpolarization. GFP‐positive rods derived from induced pluripotent stem (iPS) cells also display similar membrane current Ih as native developing rod photoreceptors, express rod‐specific phototransduction genes, and HCN‐1 channels. We conclude that Nrl‐promoter‐driven GFP‐tagged donor photoreceptors exhibit physiological characteristics of rods and that iPS cell‐derived rods in vitro may provide a renewable source for cell‐replacement therapy. STEM Cells 2013;31:1149–1159

Deletion of aryl hydrocarbon receptor AHR in mice leads to subretinal accumulation of microglia and RPE atrophy.

PURPOSE The aryl hydrocarbon receptor (AHR) is a ligand-activated nuclear receptor that regulates cellular response to environmental signals, including UV and blue wavelength light. This study was undertaken to elucidate AHR function in retinal homeostasis. METHODS RNA-seq data sets were examined for Ahr expression in the mouse retina and rod photoreceptors. The Ahr(-/-) mice were evaluated by fundus imaging, optical coherence tomography, histology, immunohistochemistry, and ERG. For light damage experiments, adult mice were exposed to 14,000 to 15,000 lux of diffuse white light for 2 hours. RESULTS In mouse retina, Ahr transcripts were upregulated during development, with continued increase in aging rod photoreceptors. Fundus examination of 3-month-old Ahr(-/-) mice revealed subretinal autofluorescent spots, which increased in number with age and following acute light exposure. Ahr(-/-) retina also showed subretinal microglia accumulation that correlated with autofluorescence changes, RPE abnormalities, and reactivity against immunoglobulin, complement factor H, and glial fibrillary acidic protein. Functionally, Ahr(-/-) mice displayed reduced ERG c-wave amplitudes. CONCLUSIONS The Ahr(-/-) mice exhibited subretinal accumulation of microglia and focal RPE atrophy, phenotypes observed in AMD. Together with a recently published report on another Ahr(-/-) mouse model, our study suggests that AHR has a protective role in the retina as an environmental stress sensor. As such, its altered function may contribute to human AMD progression and provide a target for pharmacological intervention.

NRL-Regulated Transcriptome Dynamics of Developing Rod Photoreceptors.

Gene regulatory networks (GRNs) guiding differentiation of cell types and cell assemblies in the nervous system are poorly understood because of inherent complexities and interdependence of signaling pathways. Here, we report transcriptome dynamics of differentiating rod photoreceptors in the mammalian retina. Given that the transcription factor NRL determines rod cell fate, we performed expression profiling of developing NRL-positive (rods) and NRL-negative (S-cone-like) mouse photoreceptors. We identified a large-scale, sharp transition in the transcriptome landscape between postnatal days 6 and 10 concordant with rod morphogenesis. Rod-specific temporal DNA methylation corroborated gene expression patterns. De novo assembly and alternative splicing analyses revealed previously unannotated rod-enriched transcripts and the role of NRL in transcript maturation. Furthermore, we defined the relationship of NRL with other transcriptional regulators and downstream cognate effectors. Our studies provide the framework for comprehensive system-level analysis of the GRN underlying the development of a single sensory neuron, the rod photoreceptor.

Distinct Signature of Altered Homeostasis in Aging Rod Photoreceptors: Implications for Retinal Diseases

Background Advanced age contributes to clinical manifestations of many retinopathies and represents a major risk factor for age-related macular degeneration, a leading cause of visual impairment and blindness in the elderly. Rod photoreceptors are especially vulnerable to genetic defects and changes in microenvironment, and are among the first neurons to die in normal aging and in many retinal degenerative diseases. The molecular mechanisms underlying rod photoreceptor vulnerability and potential biomarkers of the aging process in this highly specialized cell type are unknown. Methodology/Principal Findings To discover aging-associated adaptations that may influence rod function, we have generated gene expression profiles of purified rod photoreceptors from mouse retina at young adult to early stages of aging (1.5, 5, and 12 month old mice). We identified 375 genes that showed differential expression in rods from 5 and 12 month old mouse retina compared to that of 1.5 month old retina. Quantitative RT-PCR experiments validated expression change for a majority of the 25 genes that were examined. Macroanalysis of differentially expressed genes using gene class testing and protein interaction networks revealed overrepresentation of cellular pathways that are potentially photoreceptor-specific (angiogenesis and lipid/retinoid metabolism), in addition to age-related pathways previously described in several tissue types (oxidative phosphorylation, stress and immune response). Conclusions/Significance Our study suggests a progressive shift in cellular homeostasis that may underlie aging-associated functional decline in rod photoreceptors and contribute to a more permissive state for pathological processes involved in retinal diseases.

Treatment Paradigms for Retinal and Macular Diseases Using 3-D Retina Cultures Derived From Human Reporter Pluripotent Stem Cell Lines

We discuss the use of pluripotent stem cell lines carrying fluorescent reporters driven by retinal promoters to derive three-dimensional (3-D) retina in culture and how this system can be exploited for elucidating human retinal biology, creating disease models in a dish, and designing targeted drug screens for retinal and macular degeneration. Furthermore, we realize that stem cell investigations are labor-intensive and require extensive resources. To expedite scientific discovery by sharing of resources and to avoid duplication of efforts, we propose the formation of a Retinal Stem Cell Consortium. In the field of vision, such collaborative approaches have been enormously successful in elucidating genetic susceptibility associated with age-related macular degeneration.

Decreased density of lymphocyte benzodiazepine receptors in drug-resistant epileptic patients

Peripheral benzodiazepine receptors (PBR) may have a role in epilepsy and in mediating antiepileptic drug effects. Since PBR in blood mononuclear cells may be acted on by anticonvulsant drugs, we investigated possible modifications of these receptors in newly diagnosed patients, before and after antiepileptic treatment and in drug-resistant epileptic patients. A significantly lower receptor density, with no difference in affinity, was observed in drug-resistant patients, compared to newly diagnosed patients and to normal age-related controls. We suggest the possible use of PBR as a peripheral marker of drug response.

stem cells and neuronal repair

For the longest time, neurology, embryology and related textbooks have testified that development of the central nervous system (CNS) in humans is complete shortly after birth and that neuronal damage cannot be repaired endogenously since no cell proliferation is present and no new neurogenesis occurs in the mature brain. Nonetheless, early in 20th century evidence began to accumulate, from anatomical and autoradiographic studies, that mitotic activity in the nervous system persists into adult life, especially in the subependymal layer of experimental rodents. Hypotheses were proposed in the 1940 and 1950 that newly-formed cells might migrate from their site of origin into other brain areas and give rise to new neurons and glia. A decade later, H -thymidine incorporation was demonstrated in the spinal cord, hippocampus, subependymal layer and olfactory bulb in mouse and rat suggestive of DNA replication and cell division. It became clear that a majority of newly postmitotic cells from the subependymal layer migrate to the olfactory bulb, with small proportions migrating to the anterior neocortex and basal ganglia in young adult rats. However, it was not until the mid 1990s that the concept of neurogenesis in the adult central nervous system and the possibility for its regeneration was embraced and the textbooks re-written. A few years earlier, the first neural stem cell lines had been generated and propagated in vitro from fetal brain tissue. doi : 10.5214/ans.0972.7531.2009.160401

Minimal Efficacy of Nitisinone Treatment in a Novel Mouse Model of Oculocutaneous Albinism, Type 3

Purpose Oral nitisinone has been shown to increase fur and ocular pigmentation in a mouse model of oculocutaneous albinism (OCA) due to hypomorphic mutations in tyrosinase (TYR), OCA1B. This study determines if nitisinone can improve ocular and/or fur pigmentation in a mouse model of OCA type 3 (OCA3), caused by mutation of the tyrosinase-related protein 1 (Tyrp1) gene. Methods Mice homozygous for a null allele in the Tyrp1 gene (C57BL/6J-Tyrp1 b-J/J) were treated with 8 mg/kg nitisinone or vehicle every other day by oral gavage. Changes in fur and ocular melanin pigmentation were monitored. Mature ocular melanosome number and size were quantified in pigmented ocular structures by electron microscopy. Results C57BL/6J-Tyrp1 b-J/J mice carry a novel c.403T>A; 404delG mutation in Tyrp1, predicted to result in premature truncation of the TYRP1 protein. Nitisinone treatment resulted in an approximately 7-fold increase in plasma tyrosine concentrations without overt toxicity. After 1 month of treatment, no change in the color of fur or pigmented ocular structures was observed. The distribution of melanosome cross-sectional area was unchanged in ocular tissues. There was no significant difference in the number of pigmented melanosomes in the RPE/choroid of nitisinone-treated and control groups. However, there was a significant difference in the number of pigmented melanosomes in the iris. Conclusions Treatment of a mouse model of OCA3 with oral nitisinone did not have a favorable clinical effect on melanin production and minimally affected the number of pigmented melanosomes in the iris stroma. As such, treatment of OCA3 patients with nitisinone is unlikely to be therapeutic.