Defects of full-length dystrophin trigger retinal neuron damage and synapse alterations by disrupting functional autophagy

Abstract

Dystrophin (dys) mutations predispose Duchenne muscular disease (DMD) patients to brain and retinal complications. Although different dys variants, including long dys products, are expressed in the retina, their function is largely unknown. We investigated the putative role of full-length dystrophin in the homeostasis of neuro-retina and its impact on synapsis stabilization and cell fate. Retinas of mdx mice, the most used DMD model which does not express the 427-KDa dys protein (Dp427), showed overlapped cell death and impaired autophagy. Apoptotic neurons in the outer plexiform/inner nuclear layer and the ganglion cell layer had an impaired autophagy with accumulated autophagosomes. The autophagy dysfunction localized at photoreceptor axonal terminals and bipolar, amacrine, and ganglion cells. The absence of Dp427 does not cause a severe phenotype but alters the neuronal architecture, compromising mainly the pre-synaptic photoreceptor terminals and their post-synaptic sites. The analysis of two dystrophic mutants of the fruit fly Drosophila melanogaster, the homozygous Dys E17 and Dys EP3397 , lacking functional large-isoforms of dystrophin-like protein, revealed rhabdomere degeneration. Structural damages were evident in the internal network of retina/lamina where photoreceptors make the first synapse. Both accumulated autophagosomes and apoptotic features were detected and the visual system was functionally impaired. The reactivation of the autophagosome turnover by rapamycin prevented neuronal cell death and structural changes of mutant flies and, of interest, sustained autophagy ameliorated their response to light. Overall, these findings indicate that functional full-length dystrophin is required for synapsis stabilization and neuronal survival of the retina, allowing also proper autophagy as a prerequisite for physiological cell fate and visual properties.

Nutraceutical Strategy to Counteract Eye Neurodegeneration and Oxidative Stress in Drosophila melanogaster Fed with High-Sugar Diet

“…All experiments were performed with female and male adult D. melanogaster (Oregon-R strain from Bloomington Drosophila Stock Center, Indiana University Bloomington, IN, USA). As previously reported [ 20 , 22 , 35 ], flies were routinely raised on a standard cornmeal agar food (pH 5.5) at 25 °C with minor modifications. Fly food was prepared as follows: 100 g of yellow cornmeal, 100 g of brewer’s yeast, 8 g of agar, and 75 g of sucrose (5% w / v ) were mixed and dissolved by adding warm plain water to a final volume of 1.5 l, the hydration source of the flies.…”

“…Therefore, subsequent experiments were performed using Lisosan G at a final concentration of 10 µg/mL. Where indicated, flies were also reared on a high-sucrose diet supplemented with 10 nM rapamycin as previously reported [ 20 ], both in the absence and in the presence of Lisosan G.…”

“…Using published protocols [ 20 , 22 , 35 , 36 ], D. melanogaster heads were immersion-fixed overnight or for 48 h, in 4% paraformaldehyde in 0.1 M Phosphate Buffer (PB) at 4 °C, transferred to 12% sucrose in PB and stored at 4 °C for at least 24 h. Longitudinal and cross sections (10 µm) were obtained by a cryostat, mounted onto positively charged slides and stored at −20 °C until use. To allow proper comparison in the retina/eye, the same depth/region of the structure was sectioned.…”

“…For immunostaining detection, longitudinal sections were washed in PB and then pre-incubated for 30 min at room temperature with 5% BSA and 10% of normal goat serum in PB containing 0.5% Triton X-100. Pre-treated sections were incubated overnight at 4 °C with one of the following rabbit primary antibodies: anti-nitrotyrosine (1:100), anti-cleaved caspase 3 (1:500), anti-LC3 (1:100), and anti-p62/SQSTM1 (1:200) [ 20 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 ] in PB containing 0.5% Triton X-100. Following washes in PB, the sections were incubated in the appropriate Alexa Fluor secondary antibodies (1:200) in PB for 1.5 h at room temperature.…”

“…Although the mechanisms by which ROS influence neuronal damage in DR are still not fully understood [ 4 , 5 , 6 ], the most important histological feature during hyperglycemia and DR at early stages is neuronal apoptosis [ 7 , 8 , 9 ]. Alterations of the catabolic pathway autophagy also play a key role in different retina dysfunctions, including DR [ 5 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 ]. Recently, we used the fruit fly Drosophila melanogaster fed with high-sucrose diets as an in vivo model of hyperglycemia-induced neuronal damage [ 22 ].…”