R. Barhoum

Functional and structural modifications during retinal degeneration in the rd10 mouse

Mouse models of retinal degeneration are useful tools to study therapeutic approaches for patients affected by hereditary retinal dystrophies. We have studied degeneration in the rd10 mice both by immunocytochemistry and TUNEL-labeling of retinal cells, and through electrophysiological recordings. The cell degeneration in the retina of rd10 mice produced appreciable morphological changes in rod and cone cells by P20. Retinal cell death is clearly observed in the central retina and it peaked at P25 when there were 800 TUNEL-positive cells per mm(2). In the central retina, only one row of photoreceptors remained in the outer nuclear layer by P40 and there was a remarkable deterioration of bipolar cell dendrites postsynaptic to photoreceptors. The axon terminals of bipolar cells also underwent atrophy and the inner retina was subject to further changes, including a reduction and disorganization of AII amacrine cell population. Glutamate sensitivity was tested in rod bipolar cells with the single cell patch-clamp technique in slice preparations, although at P60 no significant differences were observed with age-matched controls. Thus, we conclude that rod and cone degeneration in the rd10 mouse model is followed by deterioration of their postsynaptic cells and the cells in the inner retina. However, the functional preservation of receptors for photoreceptor transmission in bipolar cells may open new therapeutic possibilities.

The protein kinase DYRK1A regulates caspase-9-mediated apoptosis during retina development.

The precise regulation of programmed cell death is critical for the normal development of the nervous system. We show here that DYRK1A (minibrain), a protein kinase essential for normal growth, is a negative regulator of the intrinsic apoptotic pathway in the developing retina. We provide evidence that changes in Dyrk1A gene dosage in the mouse strongly alter the cellularity of inner retina layers and result in severe functional alterations. We show that DYRK1A does not affect the proliferation or specification of retina progenitor cells, but rather regulates the number of cells that die by apoptosis. We demonstrate that DYRK1A phosphorylates caspase-9 on threonine residue 125, and that this phosphorylation event is crucial to protect retina cells from apoptotic cell death. Our data suggest a model in which dysregulation of the apoptotic response in differentiating neurons participates in the neuropathology of diseases that display DYRK1A gene-dosage imbalance effects, such as Downs syndrome.

Attenuation of Vision Loss and Delay in Apoptosis of Photoreceptors Induced by Proinsulin in a Mouse Model of Retinitis Pigmentosa

PURPOSE Retinitis pigmentosa (RP) is a heterogeneous group of inherited conditions that lead to blindness and for which there is no effective therapy. Apoptosis of photoreceptors is a common feature in animal models of the disease. Thus, the authors studied the therapeutic potential of proinsulin, an antiapoptotic molecule active during retinal development. METHODS Transgenic mice expressing human proinsulin (hPi) in the skeletal muscle were generated in a mixed C57BL/6:SJL background and were back-crossed to a C57BL/6 background. Two independent lineages of transgenic mice were established in which hPi production in muscle was constitutive and not regulated by glucose levels. hPi levels in serum, muscle, and retina were determined with a commercial ELISA kit, visual function was evaluated by electroretinographic (ERG) recording, and programmed cell death was assessed by TUNEL. Immunohistochemistry was used to evaluate retinal structure preservation and oxidative damage. RESULTS Transgenic expression of hPi in the rd10 retinal degeneration mouse model led to prolonged vision, as determined by ERG recording, in a manner that was related to the level of transgene expression. This attenuation of visual deterioration was correlated with a delay in photoreceptor apoptosis and with the preservation of retinal cytoarchitecture, particularly that of the cones. CONCLUSIONS These results provide a new basis for possible therapies to counteract retinitis pigmentosa and a new tool to characterize the mechanisms involved in the progress of retinal neurodegeneration.

RasGRF1 disruption causes retinal photoreception defects and associated transcriptomic alterations

RasGRF1 null mutant mice display impaired memory/learning and their hippocampus transcriptomic pattern includes a number of differentially expressed genes playing significant roles in sensory development and function. Odour avoidance and auditory brainstem response tests yielded normal results but electroretinographic analysis showed severe light perception impairment in the RasGRF1 knockouts. Whereas no structural alterations distinguished the retinas of wild‐type and knockout mice, microarray transcriptional analysis identified at least 44 differentially expressed genes in the retinas of these Knockout animals. Among these, Crb1, Pttg1, Folh1 and Myo7a have been previously related to syndromes involving retina degeneration. Interestingly, over‐expression of Folh1 would be expected to result in accumulation of its enzymatic product N‐acetyl‐aspartate, an event known to be linked to Canavan disease, a human cerebral degenerative syndrome often involving blindness and hearing loss. Consistently, in vivo brain nuclear magnetic resonance spectroscopy identified higher levels of N‐acetyl‐aspartate in our RasGRF1−/− mice and immunohistochemical analysis detected reduced levels of aspartoacylase, the enzyme which degrades N‐acetyl‐aspartate. These studies demonstrate for the first time the functional relevance of Ras signalling in mammalian photoreception and warrant further analysis of RasGRF1 Knockout mice as potential models to analyse molecular mechanisms underlying defective photoreception human diseases.