DRAM2: on the crossroad between trans-Golgi network and lysosomes?

Background: Autophagy is a key homeostatic process where damaged cellular components are sequestered into autophagosomes and degraded by the lysosomes. Impairment of autophagy has been implicated in inflammatory, neurodegenerative and age-related diseases including age related macular degeneration (AMD). Bi-allelic mutations in the DNA damage regulated autophagy modulator 2 (DRAM2) result in development of retinal dystrophy with early macular cone photoreceptor involvement. To better understand the role of autophagy in retinal cell function, we have generated induced pluripotent stem cells (iPSCs) from two patients with different mutations in the DRAM2 gene. Correction of these DRAM2 mutations using CRISPR/Cas9 in situ gene editing techniques yielded patient-specific isogenic iPSCs. Side by side comparison of DRAM2 mutated and isogenic control photoreceptors and RPE cells demonstrated a profound loss of DRAM2 protein and a significant downregulation of key lysosomal hydrolases (CTSD, NPC2, TPP1, PPT1) that are involved in the late stages of autophagy by breaking down proteins, lipids and associated sugars. In turn, lipid accumulation and loss of RPE and photoreceptor viability were revealed by transmission electron microscopy and lipidomic analyses. Together these data have led us to hypothesise that: 1) DRAM2 is critically involved in vesicular transport of key lysosomal enzymes from the trans-Golgi network to the lysosomes; 2) DRAM2 deficiency/dysfunction results in impaired transport of lysosomal enzymes which in turn causes lysosome dysfunction and lipid/waste accumulation in photoreceptors and RPE cells; 3) unprocessed waste accumulation causes progressive photoreceptor and RPE cell loss.
Building upon these pilot data, we here propose to 1) assess DRAM2 processing and trafficking in photoreceptors and RPE cells; 2) dissect the DRAM2-associated functional module; and 3) test the effect of DRAM2 supplementation in abolishing lipid and waste accumulation in photoreceptors and RPE cells.
Experimental approach: In vitro culture and differentiation of DRAM2 iPSCs and isogenic controls to photoreceptors and RPE cells using protocols established in our groups. Yeast two hybrid combined with site directed mutagenesis and directed co-immunoprecipitation assays will be used to identify and validate DRAM2-interacting proteins. Tagging of endogenous DRAM2 with a split-GFP system combined with confocal microscopy and differential centrifugation and density-gradient dependent organelle isolation will be used to address DRAM2 processing and trafficking through the endoplasmic reticulum and trans-Golgi network to late endosomes and lysosomes. Importantly AAV based supplementation of DRAM2 will be tested to restore expression of functional DRAM2 in photoreceptor and RPE cells to reverse waste accumulation and loss of viability.