Gene therapy regenerates protein expression in cone photoreceptors in Rpe65(R91W/R91W) mice.

Cone photoreceptors mediate visual acuity under daylight conditions, so loss of cone-mediated central vision of course dramatically affects the quality of life of patients suffering from retinal degeneration. Therefore, promoting cone survival has become the goal of many ocular therapies and defining the stage of degeneration that still allows cell rescue is of prime importance. Using the Rpe65(R91W/R91W) mouse, which carries a mutation in the Rpe65 gene leading to progressive photoreceptor degeneration in both patients and mice, we defined stages of retinal degeneration that still allow cone rescue. We evaluated the therapeutic window within which cones can be rescued, using a subretinal injection of a lentiviral vector driving expression of RPE65 in the Rpe65(R91W/R91W) mice. Surprisingly, when applied to adult mice (1 month) this treatment not only stalls or slows cone degeneration but, actually, induces cone-specific protein expression that was previously absent. Before the intervention only part of the cones (40% of the number found in wild-type animals) in the Rpe65(R91W/R91W) mice expressed cone transducin (GNAT2); this fraction increased to 64% after treatment. Correct S-opsin localization is also recovered in the transduced region. In consequence these results represent an extended therapeutic window compared to the Rpe65(-/-) mice, implying that patients suffering from missense mutations might also benefit from a prolonged therapeutic window. Moreover, cones are not only rescued during the course of the degeneration, but can actually recover their initial status, meaning that a proportion of altered cones in chromophore deficiency-related disease can be rehabilitated even though they are severely affected.

Remaining rod activity mediates visual behavior in adult Rpe65-/- mice.

PURPOSE: C57/Bl6, Cpfl1(-/-) (cone photoreceptors function loss 1; pure rod function), Gnat1a(-/-) (rod α-transducin; pure cone function), and Rpe65(-/-);Rho(-/-) double-knockout mice were studied to distinguish the respective contributions of the different photoreceptor (PR) systems that enable light perception and mediate a visual reflex in adult Rpe65(-/-) mice, with a simple behavioral procedure. METHODS: Visual function was estimated using a rotating automated optomotor drum covered with vertical black-and-white stripes at spatial frequencies of 0.025 to 0.5 cycles per degree (cyc/deg) in both photopic and scotopic conditions. Mouse strains with different luminances were tested to evaluate the contribution and the light-intensity threshold of each PR system. RESULTS: Stripe rotation elicited head movements in the wild-type (WT) animals in photopic and scotopic conditions, depending on the spatial frequency, whereas the Cpfl1(-/-) mice show a reduced activity in the photopic condition and the Gnat1a(-/-) mice an almost absent response in the scotopic condition. A robust visual response was obtained with Rpe65(-/-) knockout mice at 0.075 and 0.1 cyc/deg in the photopic condition. The residual rod function in the Rpe65(-/-) animals was demonstrated by testing Rpe65(-/-);Rho(-/-) mice that present no response in photopic conditions. CONCLUSIONS: The optomotor test is a simple method of estimating the visual function and evaluating the respective contributions of rod and cone systems. This test was used to demonstrate that in Rpe65(-/-) mice, devoid of functional cones and of detectable 11-cis-retinal protein, the rods mimic cone function in part, by mediating vision in photopic conditions.

Dissecting the mechanisms of tissue transglutaminase-induced cross-linking of alpha-synuclein: implications for the pathogenesis of Parkinson disease.

Tissue transglutaminase (tTG) has been implicated in the pathogenesis of Parkinson disease (PD). However, exactly how tTG modulates the structural and functional properties of alpha-synuclein (alpha-syn) and contributes to the pathogenesis of PD remains unknown. Using site-directed mutagenesis combined with detailed biophysical and mass spectrometry analyses, we sought to identify the exact residues involved in tTG-catalyzed cross-linking of wild-type alpha-syn and alpha-syn mutants associated with PD. To better understand the structural consequences of each cross-linking reaction, we determined the effect of tTG-catalyzed cross-linking on the oligomerization, fibrillization, and membrane binding of alpha-syn in vitro. Our findings show that tTG-catalyzed cross-linking of monomeric alpha-syn involves multiple cross-links (specifically 2-3). We subjected tTG-catalyzed cross-linked monomeric alpha-syn composed of either wild-type or Gln --> Asn mutants to sequential proteolysis by multiple enzymes and peptide mapping by mass spectrometry. Using this approach, we identified the glutamine and lysine residues involved in tTG-catalyzed intramolecular cross-linking of alpha-syn. These studies demonstrate for the first time that Gln(79) and Gln(109) serve as the primary tTG reactive sites. Mutating both residues to asparagine abolishes tTG-catalyzed cross-linking of alpha-syn and tTG-induced inhibition of alpha-syn fibrillization in vitro. To further elucidate the sequence and structural basis underlying these effects, we identified the lysine residues that form isopeptide bonds with Gln(79) and Gln(109). This study provides mechanistic insight into the sequence and structural basis of the inhibitory effects of tTG on alpha-syn fibrillogenesis in vivo, and it sheds light on the potential role of tTG cross-linking on modulating the physiological and pathogenic properties of alpha-syn.