Rer1-mediated quality control system is required for neural stem cell maintenance during cerebral cortex development.

Rer1 is a retrieval receptor for endoplasmic reticulum (ER) retention of various ER membrane proteins and unassembled or immature components of membrane protein complexes. However, its physiological functions during mammalian development remain unclear. This study aimed to investigate the role of Rer1-mediated quality control system in mammalian development. We show that Rer1 is required for the sufficient cell surface expression and activity of γ-secretase complex, which modulates Notch signaling during mouse cerebral cortex development. When Rer1 was depleted in the mouse cerebral cortex, the number of neural stem cells decreased significantly, and malformation of the cerebral cortex was observed. Rer1 loss reduced γ-secretase activity and downregulated Notch signaling in the developing cerebral cortex. In Rer1-deficient cells, a subpopulation of γ-secretase complexes and components was transported to and degraded in lysosomes, thereby significantly reducing the amount of γ-secretase complex on the cell surface. These results suggest that Rer1 maintains Notch signaling by maintaining sufficient expression of the γ-secretase complex on the cell surface and regulating neural stem cell maintenance during cerebral cortex development.

Autophagy sequesters damaged lysosomes to control lysosomal biogenesis and kidney injury.

Diverse causes, including pathogenic invasion or the uptake of mineral crystals such as silica and monosodium urate (MSU), threaten cells with lysosomal rupture, which can lead to oxidative stress, inflammation, and apoptosis or necrosis. Here, we demonstrate that lysosomes are selectively sequestered by autophagy, when damaged by MSU, silica, or the lysosomotropic reagent L-Leucyl-L-leucine methyl ester (LLOMe). Autophagic machinery is recruited only on damaged lysosomes, which are then engulfed by autophagosomes. In an autophagy-dependent manner, low pH and degradation capacity of damaged lysosomes are recovered. Under conditions of lysosomal damage, loss of autophagy causes inhibition of lysosomal biogenesis in vitro and deterioration of acute kidney injury in vivo. Thus, we propose that sequestration of damaged lysosomes by autophagy is indispensable for cellular and tissue homeostasis.

Selective autophagy: lysophagy.

Autophagy is a bulk degradation system that is induced under stress conditions such as nutrient deprivation. Selective autophagy, including xenophagy and mitophagy, is believed to play important roles in the development of several diseases. Consequently, selective autophagy represents a potential therapeutic target. Recent work showed that the lysosome, a membrane-bound acidic organelle, is selectively sequestered by autophagy when its membrane is injured; this phenomenon is called "lysophagy". Lysosomes can be injured by diverse causes, including amyloid proteins and mineral crystals such as silica and monosodium urate, which would trigger neurodegeneration and other diseases. In this section, we provide an overview of methods for monitoring lysophagy in mammalian cultured cells. These methods can be used to evaluate the involvement of molecules of interest in selective autophagy, and in screens aimed at identifying novel proteins engaged in selective autophagy.

RAB35 is required for murine hippocampal development and functions by regulating neuronal cell distribution.

RAB35 is a multifunctional small GTPase that regulates endocytic recycling, cytoskeletal rearrangement, and cytokinesis. However, its physiological functions in mammalian development remain unclear. Here, we generated Rab35-knockout mice and found that RAB35 is essential for early embryogenesis. Interestingly, brain-specific Rab35-knockout mice displayed severe defects in hippocampal lamination owing to impaired distribution of pyramidal neurons, although defects in cerebral cortex formation were not evident. In addition, Rab35-knockout mice exhibited defects in spatial memory and anxiety-related behaviors. Quantitative proteomics indicated that the loss of RAB35 significantly affected the levels of other RAB proteins associated with endocytic trafficking, as well as some neural cell adhesion molecules, such as contactin-2. Collectively, our findings revealed that RAB35 is required for precise neuronal distribution in the developing hippocampus by regulating the expression of cell adhesion molecules, thereby influencing spatial memory.

Autophagy-targeted therapy to modulate age-related diseases: Success, pitfalls, and new directions.

Autophagy is a critical metabolic process that supports homeostasis at a basal level and is dynamically regulated in response to various physiological and pathological processes. Autophagy has some etiologic implications that support certain pathological processes due to alterations in the lysosomal-degradative pathway. Some of the conditions related to autophagy play key roles in highly relevant human diseases, e.g., cardiovascular diseases (15.5%), malignant and other neoplasms (9.4%), and neurodegenerative conditions (3.7%). Despite advances in the discovery of new strategies to treat these age-related diseases, autophagy has emerged as a therapeutic option after preclinical and clinical studies. Here, we discuss the pitfalls and success in regulating autophagy initiation and its lysosome-dependent pathway to restore its homeostatic role and mediate therapeutic effects for cancer, neurodegenerative, and cardiac diseases. The main challenge for the development of autophagy regulators for clinical application is the lack of specificity of the repurposed drugs, due to the low pharmacological uniqueness of their target, including those that target the PI3K/AKT/mTOR and AMPK pathway. Then, future efforts must be conducted to deal with this scenery, including the disclosure of key components in the autophagy machinery that may intervene in its therapeutic regulation. Among all efforts, those focusing on the development of novel allosteric inhibitors against autophagy inducers, as well as those targeting autolysosomal function, and their integration into therapeutic regimens should remain a priority for the field.

Autophagy Inhibits the Accumulation of Advanced Glycation End Products by Promoting Lysosomal Biogenesis and Function in the Kidney Proximal Tubules.

Advanced glycation end products (AGEs) are involved in the progression of diabetic nephropathy. AGEs filtered by glomeruli or delivered from the circulation are endocytosed and degraded in the lysosomes of kidney proximal tubular epithelial cells (PTECs). Autophagy is a highly conserved degradation system that regulates intracellular homeostasis by engulfing cytoplasmic components. We have recently demonstrated that autophagic degradation of damaged lysosomes is indispensable for cellular homeostasis in some settings. In this study, we tested the hypothesis that autophagy could contribute to the degradation of AGEs in the diabetic kidney by modulating lysosomal biogenesis. Both a high-glucose and exogenous AGE overload gradually blunted autophagic flux in the cultured PTECs. AGE overload upregulated lysosomal biogenesis and function in vitro, which was inhibited in autophagy-deficient PTECs because of the impaired nuclear translocation of transcription factor EB. Consistently, streptozotocin-treated, PTEC-specific, autophagy-deficient mice failed to upregulate lysosomal biogenesis and exhibited the accumulation of AGEs in the glomeruli and renal vasculature as well as in the PTECs, along with worsened inflammation and fibrosis. These results indicate that autophagy contributes to the degradation of AGEs by the upregulation of lysosomal biogenesis and function in diabetic nephropathy. Strategies aimed at promoting lysosomal function hold promise for treating diabetic nephropathy.

Rer1p regulates the ER retention of immature rhodopsin and modulates its intracellular trafficking.

Rhodopsin is a pigment in photoreceptor cells. Some rhodopsin mutations cause the protein to accumulate in the endoplasmic reticulum (ER), leading to photoreceptor degeneration. Although several mutations have been reported, how mutant rhodopsin is retained in the ER remains unclear. In this study, we identified Rer1p as a modulator of ER retention and rhodopsin trafficking. Loss of Rer1p increased the transport of wild-type rhodopsin to post-Golgi compartments. Overexpression of Rer1p caused immature wild-type rhodopsin to accumulate in the ER. Interestingly, the G51R rhodopsin mutant, which has a mutation in the first transmembrane domain and accumulates in the ER, was released to the plasma membrane or lysosomes in Rer1-knockdown cells. Consistent with these results, Rer1p interacted with both wild-type and mutant rhodopsin. These results suggest that Rer1p regulates the ER retention of immature or misfolded rhodopsin and modulates its intracellular trafficking through the early secretory pathway.

Two Beclin 1-binding proteins, Atg14L and Rubicon, reciprocally regulate autophagy at different stages.

Beclin 1, a protein essential for autophagy, binds to hVps34/Class III phosphatidylinositol-3-kinase and UVRAG. Here, we have identified two Beclin 1 associated proteins, Atg14L and Rubicon. Atg14L and UVRAG bind to Beclin 1 in a mutually exclusive manner, whereas Rubicon binds only to a subpopulation of UVRAG complexes; thus, three different Beclin 1 complexes exist. GFP-Atg14L localized to the isolation membrane and autophagosome, as well as to the ER and unknown puncta. Knockout of Atg14L in mouse ES cells caused a defect in autophagosome formation. GFP-Rubicon was localized at the endosome/lysosome. Knockdown of Rubicon caused enhancement of autophagy, especially at the maturation step, as well as enhancement of endocytic trafficking. These data suggest that the Beclin 1-hVps34 complex functions in two different steps of autophagy by altering the subunit composition.

New aspects of a small GTPase RAB35 in brain development and function.

In eukaryotic cells, organelles in the secretory, lysosomal, and endocytic pathways actively exchange biological materials with each other through intracellular membrane trafficking, which is the process of transporting the cargo of proteins, lipids, and other molecules to appropriate compartments via transport vesicles or intermediates. These processes are strictly regulated by various small GTPases such as the RAS-like in rat brain (RAB) protein family, which is the largest subfamily of the RAS superfamily. Dysfunction of membrane trafficking affects tissue homeostasis and leads to a wide range of diseases, including neurological disorders and neurodegenerative diseases. Therefore, it is important to understand the physiological and pathological roles of RAB proteins in brain function. RAB35, a member of the RAB family, is an evolutionarily conserved protein in metazoans. A wide range of studies using cultured mammalian cells and model organisms have revealed that RAB35 mediates various processes such as cytokinesis, endocytic recycling, actin bundling, and cell migration. RAB35 is also involved in neurite outgrowth and turnover of synaptic vesicles. We generated brain-specific Rab35 knockout mice to study the physiological roles of RAB35 in brain development and function. These mice exhibited defects in anxiety-related behaviors and spatial memory. Strikingly, RAB35 is required for the precise positioning of pyramidal neurons during hippocampal development, and thereby for normal hippocampal lamination. In contrast, layer formation in the cerebral cortex occurred superficially, even in the absence of RAB35, suggesting a predominant role for RAB35 in hippocampal development rather than in cerebral cortex development. Recent studies have suggested an association between RAB35 and neurodegenerative diseases, including Parkinson's disease and Alzheimer's disease. In this review, we provide an overview of the current understanding of subcellular functions of RAB35. We also provide insights into the physiological role of RAB35 in mammalian brain development and function, and discuss the involvement of RAB35 dysfunction in neurodegenerative diseases.