Pattern of retinal morphological and functional decay in a light-inducible, rhodopsin mutant mouse

Hallmarks of Retinitis Pigmentosa (RP), a family of genetic diseases, are a typical rod-cone-degeneration with initial night blindness and loss of peripheral vision, followed by decreased daylight sight and progressive visual acuity loss up to legal blindness. Great heterogeneity in nature and function of mutated genes, variety of mutations for each of them, variability in phenotypic appearance and transmission modality contribute to make RP a still incurable disease. Translational research relies on appropriate animal models mimicking the genetic and phenotypic diversity of the human pathology. Here, we provide a systematic, morphological and functional analysis of RhoTvrm4/Rho+ rhodopsin mutant mice, originally described in 2010 and portraying several features of common forms of autosomal dominant RP caused by gain-of-function mutations. These mice undergo photoreceptor degeneration only when exposed briefly to strong, white light and allow controlled timing of induction of rod and cone death, which therefore can be elicited in adult animals, as observed in human RP. The option to control severity and retinal extent of the phenotype by regulating intensity and duration of the inducing light opens possibilities to exploit this model for multiple experimental purposes. Altogether, the unique features of this mutant make it an excellent resource for retinal degeneration research.


Inner retinal morphological and functional changes
Tvrm4 mutants mimic faithfully dominant RP and can be used to study mechanisms and pathways of cell death associated to mutations of RHO as well as the occurrence of inner retinal remodeling. Because Tvrm4 mice of the present study are adult, we investigated whether the hallmarks of phase 1 remodeling observed in other forms of retinal degeneration (i.e. rd1 mice), in which phenotype onset overlaps to the late retinal synaptogenesis, also occur in the novel mutant.
We studied the morphology of the dendrites of bipolar and horizontal cells in the OPL and examined the morphology of the innermost retinal neurons in Tvrm4 mutants exposed to 12,000 lux for 1 min after 7, 14 and 21 days. We observed a significant shedding of dendritic arborization from rod bipolar cells, topographically associated with the process of rod degeneration (Figures S1, S2). Dendritic retraction is paralleled by decreased expression and mislocalization of mGluR6 receptors in rod bipolar cells; mGluR6 immunoreactive puncta are reduced in number and become misplaced along the axons of rod bipolar cells in the IPL ( Figure S2B). Dendritic retraction is clearly detectable 7 days post induction at retinal locations with prominent rod loss and ONL rows lower than 6. mGluR6 decrement and misplacement is an early indicator of OPL abnormality in the central retina, detectable 48 hours post light induction.
In parallel to rod bipolar remodeling, horizontal cells begin to show hypertrophic bodies; both dendrites and axonal endings exhibit major discontinuities in their spatial arrangement and decrement in the complexity of their process network, clearly perceivable in retinal whole mounts (Figures S1C, D and S2E, F).
Particularly evident are the changes of axonal arborizations (postsynaptic to rod terminals), which decrease in density but become thicker and spatially irregular. These abnormalities occur inside the area irradiated by the inducing light and match the zone of photoreceptor death. Sprouting of second order neurons, described in other mutants, is virtually absent here and regressive events in the outer retina predominate.
As for other rodent models, changes in specific types of cone bipolar cells are not easily detectable, for specific markers are rare. A thinning of the OPL in the retina of Tvrm4-induced mice may be appreciated when dendrites of second order neurons are stained with specific antibodies ( Figure S2C Information on the functional properties of inner retinal cells in both scotopic and photopic conditions was obtained by analyzing the oscillatory potentials (OPs) of the ERG. A significant amplitude reduction of scotopic OP1 (p=0.005, t-test), OP2 (p=0.021, t-test) and OP3 (p=0.027, t-test) is observed as early as 2 days after exposure to 12,000 lux for 1 min ( Figure S3A and C). Conversely, the photopic OPs (OP1, OP2 and OP3) are not significantly affected ( Figure S3B and D). These observations confirm the effects described above on the main ERG components and support the notion that in this animal model the primary damage starts in the rod pathway and eventually involves cones.

Dendrites of rod bipolar cells stained by PKC antibodies in whole mount retinal preparations from a control (not-induced Tvmr4) mouse (A) and a Tvrm4 mouse exposed for 1 min to 12,000 lux and observed after 7 days (B). Focus is on the OPL, which shows profusely ramified dendrites in A but cell bodies with bare and aberrant dendrites in B. C, D: same preparations as above, stained for Calbindin D (red) and neurofilament (green), showing cell bodies/dendrites and axonal compartments of horizontal cells,
respectively. Note the hypertrophic somas of horizontal cells and the loose and highly irregular meshwork of their axonal arbors.  The amplitude of OPs is significantly reduced in Tvrm4 mice exposed to steady light with respect to control animals. OPs signals are impaired as early as 2 days from phenotype induction. n=6 for each condition. Bars represent mean ± SE * P<0.05; ** P<0.01 one-way ANOVA.