Rhodopsin is a cilia-specific GPCR essential for vision. Rhodopsin mislocalization is associated with blinding diseases called retinal ciliopathies. The mechanism by which rhodopsin mislocalizes in rod photoreceptor neurons is not well understood. Therefore, we investigated the roles of trafficking signals in rhodopsin mislocalization. Rhodopsin and its truncation mutants were fused to a photoconvertible fluorescent protein, Dendra2, and expressed in Xenopus laevis rod photoreceptors. Photoconversion of Dendra2 causes a color change from green to red, enabling visualization of the dynamic events associated with rhodopsin trafficking and renewal. We found that rhodopsin mislocalization is a facilitated process for which a signal located within 322-326 aa (CCGKN) is essential. An additional signal within 327-336 aa further facilitated the mislocalization. This collective mistrafficking signal confers toxicity to rhodopsin and causes mislocalization when the VXPX cilia-targeting motif is absent. We also determined that the VXPX motif neutralizes this mistrafficking signal, enhances ciliary targeting at least 10-fold, and accelerates trafficking of post-Golgi vesicular structures. In the absence of the VXPX motif, mislocalized rhodopsin is actively cleared through secretion of vesicles into the extracellular milieu. Therefore, this study unveiled the multiple roles of trafficking signals in rhodopsin localization and renewal.

It is unclear how unconventional secretion interplays with conventional secretion for the normal maintenance and renewal of membrane structures. The photoreceptor sensory cilium is recognized for fast membrane renewal, for which rhodopsin and peripherin/rds (P/rds) play critical roles. Here, we provide evidence that P/rds is targeted to the cilia by an unconventional secretion pathway. When expressed in ciliated hTERT-RPE1 human cell line, P/rd is localized to cilia. Cilium trafficking of P/rds was sustained even when the Golgi functions, including trans-Golgi-mediated conventional secretion, were inhibited by the small molecules brefeldin A, 30N12, and monensin. The unconventional cilia targeting of P/rds is dependent on COPII-mediated exit from the ER, but appears to be independent of GRASP55-mediated secretion. The regions in the C-terminal tail of P/rds are essential for this unconventional trafficking. In the absence of the region required for cilia targeting, P/rds was prohibited from entering the secretory pathways and was retained in the Golgi apparatus. A region essential for this Golgi retention was also found in the C-terminal tail of P/rds and supported the cilia targeting of P/rds mediated by unconventional secretion. In ciliated cells, including bovine and Xenopus laevis rod photoreceptors, P/rds was robustly sensitive to endoglycosidase H, which is consistent with its bypassing the medial Golgi and traversing the unconventional secretory pathway. Because rhodopsin is known to traffic through conventional secretion, this study of P/rds suggests that both conventional secretion and unconventional secretion need to cooperate for the renewal of the photoreceptor sensory cilium.

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Predisposition to Alzheimer’s disease (AD) may arise from lipid metabolism perturbation, however, the underlying mechanism remains elusive. Here, we identify ATPase family AAA-domain containing protein 3A (ATAD3A), a mitochondrial AAA-ATPase, as a molecular switch that links cholesterol metabolism impairment to AD phenotypes. In neuronal models of AD, the 5XFAD mouse model and post-mortem AD brains, ATAD3A is oligomerized and accumulated at the mitochondria-associated ER membranes (MAMs), where it induces cholesterol accumulation by inhibiting gene expression of CYP46A1, an enzyme governing brain cholesterol clearance. ATAD3A and CYP46A1 cooperate to promote APP processing and synaptic loss. Suppressing ATAD3A oligomerization by heterozygous ATAD3A knockout or pharmacological inhibition with DA1 restores neuronal CYP46A1 levels, normalizes brain cholesterol turnover and MAM integrity, suppresses APP processing and synaptic loss, and consequently reduces AD neuropathology and cognitive deficits in AD transgenic mice. These findings reveal a role for ATAD3A oligomerization in AD pathogenesis and suggest ATAD3A as a potential therapeutic target for AD.