Disease-causing mutations in the CLRN1 gene alter normal CLRN1 protein trafficking to the plasma membrane.

Purpose: Mutations of clarin 1 (CLRN1) cause Usher syndrome type 3 (USH3). To determine the effects of USH3 mutations on CLRN1 function, we examined the cellular distribution and stability of both normal and mutant CLRN1 in vitro. We also searched for novel disease-causing mutations in a cohort of 59 unrelated Canadian and Finnish USH patients.

Severe retinal degeneration caused by a novel rhodopsin mutation.

Purpose: To identify a new mouse mutation developing early-onset dominant retinal degeneration, to determine the causative gene mutation, and to investigate the underlying mechanism.

Methods: Retinal phenotype was examined by indirect ophthalmoscopy, histology, transmission electron microscopy, immunohistochemistry, Western blot analysis, and electroretinography. Causative gene mutation was determined by genomewide linkage analysis and DNA sequencing.

CLRN1 is nonessential in the mouse retina but is required for cochlear hair cell development.

Mutations in the CLRN1 gene cause Usher syndrome type 3 (USH3), a human disease characterized by progressive blindness and deafness. Clarin 1, the protein product of CLRN1, is a four-transmembrane protein predicted to be associated with ribbon synapses of photoreceptors and cochlear hair cells, and recently demonstrated to be associated with the cytoskeleton. To study Clrn1, we created a Clrn1 knockout (KO) mouse and characterized the histological and functional consequences of Clrn1 deletion in the retina and cochlea.

Inner limiting membrane barriers to AAV-mediated retinal transduction from the vitreous.

Adeno-associated viral gene therapy has shown great promise in treating retinal disorders, with three promising clinical trials in progress. Numerous adeno-associated virus (AAV) serotypes can infect various cells of the retina when administered subretinally, but the retinal detachment accompanying this injection induces changes that negatively impact the microenvironment and survival of retinal neurons. Intravitreal administration could circumvent this problem, but only AAV2 can infect retinal cells from the vitreous, and transduction is limited to the inner retina.

Molecular evolution of adeno-associated virus for enhanced glial gene delivery.

The natural tropism of most viral vectors, including adeno-associated viral (AAV) vectors, leads to predominant transduction of neurons and epithelia within the central nervous system (CNS) and retina. Despite the clinical relevance of glia for homeostasis in neural tissue, and as causal contributors in genetic disorders such as Alzheimer's and amyotrophic lateral sclerosis, efforts to develop more efficient gene delivery vectors for glia have met with limited success. Recently, viral vector engineering involving high-throughput random diversification and selection has enabled the rapid creation of AAV vectors with valuable new gene delivery properties.

In vivo imaging of microscopic structures in the rat retina.

Purpose: The ability to resolve single retinal cells in rodents in vivo has applications in rodent models of the visual system and retinal disease. The authors have characterized the performance of a fluorescence adaptive optics scanning laser ophthalmoscope (fAOSLO) that provides cellular and subcellular imaging of rat retina in vivo.

Methods: Enhanced green fluorescent protein (eGFP) was expressed in retinal ganglion cells of normal Sprague-Dawley rats via intravitreal injections of adeno-associated viral vectors.

Clarin-1, encoded by the Usher Syndrome III causative gene, forms a membranous microdomain: possible role of clarin-1 in organizing the actin cytoskeleton.

Clarin-1 is the protein product encoded by the gene mutated in Usher syndrome III. Although the molecular function of clarin-1 is unknown, its primary structure predicts four transmembrane domains similar to a large family of membrane proteins that include tetraspanins. Here we investigated the role of clarin-1 by using heterologous expression and in vivo model systems.

Usher syndrome IIIA gene clarin-1 is essential for hair cell function and associated neural activation.

Usher syndrome 3A (USH3A) is an autosomal recessive disorder characterized by progressive loss of hearing and vision due to mutation in the clarin-1 (CLRN1) gene. Lack of an animal model has hindered our ability to understand the function of CLRN1 and the pathophysiology associated with USH3A. Here we report for the first time a mouse model for ear disease in USH3A.

Outcomes for older adults in an inpatient rehabilitation facility following hip fracture (HF) surgery.

The purpose of the study was to evaluate patient and system outcomes regarding older community-residing adults who participated in a rehabilitation program following HF surgery. The health care professionals on the rehabilitation unit in this feasibility study had never cared for such patients who were so frail, with multiple co-morbidities including cognitive impairment (CI). After an innovative model of care was developed and the staff trained in the novel approach to care, the unit opened for all patients living within the community who had fractured their hip, regardless of their CI.

Systemic aminoglycoside treatment in rodent models of retinitis pigmentosa.

We studied the potential of systemically administered aminoglycosides as a therapy for retinal degeneration resulting from premature termination codon (PTC) mutations. Aminoglycosides were systemically delivered to two rodent models of retinal degeneration: a transgenic rat model of dominant disease caused by a PTC in rhodopsin (S334ter); and a mouse model of recessive disease (rd12) caused by a PTC in the retinoid isomerase Rpe65. Initial luciferase reporter assays were undertaken to measure the efficiency of gentamicin-induced read-through in vitro.

In vitro analysis of promoter activity in Müller cells.

Purpose: Rational modification of promoter architecture is necessary for manipulation of transgene activity and requires accurate deciphering of regulatory control elements. Identification of minimally sized promoters is critical to the design of viral vectors for gene therapy. To this end, we evaluated computational methods for predicting short DNA sequences capable of driving gene expression in Müller cells.

Patient education as a strategy for provider education and engagement: a case study using myJointReplacement.ca.

MyJointReplacement.ca was initiated to integrate patient and provider perspectives with the evidence on joint replacement care into a patient education website to promote consistency in practice. The project's leadership ensured that the project fit into a larger system change initiative.

Rapid and stable knockdown of an endogenous gene in retinal pigment epithelium.

The selective silencing of target genes in specific cell types by RNA interference (RNAi) represents a powerful approach both to gene therapy of dominantly active mutant alleles, and to the investigation of normal gene function in animal models in vivo. We established a simple and versatile in vitro method for screening the efficacy of DNA-based short hairpin RNAs (shRNAs), and identified a highly effective shRNA targeting basic fibroblast growth factor (bFGF), a gene thought to play important roles in endogenous neuroprotective responses in the rat retina. We used two viral vectors, based on lentivirus and adeno-associated virus (AAV), to deliver shRNAs and silence bFGF in retinal pigment epithelial cells in vivo.

Very low density lipoprotein receptor, a negative regulator of the wnt signaling pathway and choroidal neovascularization.

Choroidal neovascularization (CNV) in age-related macular degeneration is a leading cause of blindness. Very low density lipoprotein receptor gene knock-out (Vldlr(-/-)) mice have been shown to develop subretinal neovascularization (NV) with an unknown mechanism. The present study showed that in Vldlr(-/-) mice, NV initiated in the choroid and progressed to penetrate the retinal pigment epithelium layer, proliferating in the subretinal space.

Cognitive-behavioural stimulation protocol for severely brain-damaged patients in the post-acute stage in developmental age.

Purpose: To present a cognitive-behavioural stimulation (CBS) protocol designed to help severely damaged patients in the early post-acute stage by describing the underlying methodology and assessing its efficacy compared to traditional rehabilitation methods. This protocol combines multisensory stimulation and cognitive-behavioural techniques to elicit and intensify the occurrence of adaptive responses and reduce maladaptive behavioural patterns.

Methods: A control group and an experimental group--both evaluated with the Levels of Cognitive Functioning Assessment Scale (LOCFAS)--were compared at the beginning of the rehabilitation programme and at the end of it.

Functional promoter testing using a modified lentiviral transfer vector.

Purpose: The importance of retinal glial cells in the maintenance of retinal health and in retinal degenerations has not been fully explored. Several groups have suggested that secretion of neurotrophic proteins from the retina's primary glial cell type, the Müller cell, holds promise for treating retinal degenerations. Tight regulation of transgene expression in Müller cells is likely to be critical to the efficacy of long-term neuroprotective therapies, due to the genetic heterogeneity and progressive nature of retinal disease.

Transport of truncated rhodopsin and its effects on rod function and degeneration.

Purpose: Most transgenic animal models of retinal degeneration caused by rhodopsin mutations express the rhodopsin transgene on a wild-type (WT) genetic background. Previous studies have demonstrated that one mechanism of retinal degeneration is rhodopsin overexpression. To study the effect of C-terminal truncation of rhodopsin without the confounding factors of overexpression, several lines of transgenic mice were generated that expressed a C-terminal rhodopsin mutation on rhodopsin-knockout backgrounds.

Remote control of neuronal activity with a light-gated glutamate receptor.

The ability to stimulate select neurons in isolated tissue and in living animals is important for investigating their role in circuits and behavior. We show that the engineered light-gated ionotropic glutamate receptor (LiGluR), when introduced into neurons, enables remote control of their activity. Trains of action potentials are optimally evoked and extinguished by 380 nm and 500 nm light, respectively, while intermediate wavelengths provide graded control over the amplitude of depolarization.

Anti-clarin-1 AAV-delivered ribozyme induced apoptosis in the mouse cochlea.

Usher syndrome type 3 is caused by mutations in the USH3A gene, which encodes the protein clarin-1. Clarin-1 is a member of the tetraspanin superfamily (TM4SF) of transmembrane proteins, expressed in the organ of Corti and spiral ganglion cells of the mouse ear. We have examined whether the AAV-mediated anti-clarin ribozyme delivery causes apoptotic cell death in vivo in the organ of Corti.

Key factors in back disability prevention: a consensus panel on their impact and modifiability.

Study Design: Modified Delphi panel using Q-methodology.

Objective: To reach consensus on the relative impact and modifiability of factors that might prevent participation restrictions in people with back pain as defined by the WHO International Classification of Functioning, Disability and Health.

Summary Of Background Data: Back disability prevention should focus on factors with large impact that are relatively easy to change and supported by most stakeholders.

Targeted transgene expression in muller glia of normal and diseased retinas using lentiviral vectors.

Purpose: Müller glia play crucial roles in retinal homeostasis and function. Genetic modification of Müller cells by viral gene delivery would be valuable for studies of their normal physiology and roles in retinal disease states. However, stable and efficient transgene expression in Müller cells after delivery of gene transfer vectors has remained elusive.

Characterization of peripherin/rds and rom-1 transport in rod photoreceptors of transgenic and knockout animals.

Purpose: Peripherin/rds and rom-1 have structural roles in morphogenesis and stabilization of the outer segment, but little is known about their transport and sorting to the rod outer segment. Peripherin/rds and rom-1 trafficking were studied in several knockout and transgenic animal models.

Methods: Rod outer segment formation and distribution of peripherin/rds and rom-1 were examined by immunohistochemistry, electron microscopy, and molecular biological methods in wild-type, rhodopsin-knockout, and peripherin/rds-knockout mice.

Looking within for vision.

Channelrhodopsin-2 (ChR2), a directly light-gated cation channel from the green alga Chlamydomonas reinhardtii has been shown to be a directly light-switched cation-selective ion channel, which employs 11-cis retinal as its chromophore. This is the same chromophore as the mammalian photoreceptor's visual pigment-rhodopsin. Previously, investigators demonstrated that ChR2 can be used to optically control neuronal firing by depolarizing the cell.