Defects in endolysosomal and autophagic functions are increasingly viewed as key pathological features of neurodegenerative disorders. A master regulator of these functions is phosphatidylinositol-3-phosphate (PI3P), a phospholipid synthesized primarily by class III PI 3-kinase Vps34. Here we report that disruption of neuronal Vps34 function in vitro and in vivo impairs autophagy, lysosomal degradation as well as lipid metabolism, causing endolysosomal membrane damage. PI3P deficiency also promotes secretion of unique exosomes enriched for undigested lysosomal substrates, including amyloid precursor protein C-terminal fragments (APP-CTFs), specific sphingolipids, and the phospholipid bis(monoacylglycero)phosphate (BMP), which normally resides in the internal vesicles of endolysosomes. Secretion of these exosomes requires neutral sphingomyelinase 2 and sphingolipid synthesis. Our results reveal a homeostatic response counteracting lysosomal dysfunction via secretion of atypical exosomes eliminating lysosomal waste and define exosomal APP-CTFs and BMP as candidate biomarkers for endolysosomal dysfunction associated with neurodegenerative disorders.
SignificanceMembranes can adopt distinct phases. The endoplasmic reticulum (ER) is the largest membrane system inside cells and also harbors the richest metabolic activity including lipid synthesis. Unlike plasma membrane where separated “lipid raft” domains have been predicted and observed, ER membrane is thought to be uniformly fluidic. However, such understanding is based on biophysical studies of model membrane under thermodynamic equilibrium. It remains unclear whether and how lipid synthesis activity perturbs the equilibrium and promotes phase segregation in ER membrane. Herein, we utilized coherent Raman imaging technique to track lipid synthesis and surprisingly revealed solid-like domains emerging from liquid ER membrane. Interestingly, this phenomenon can be tuned by the incoming nutrient source, demonstrating the susceptibility of ER membrane to nonequilibrium modulation.
SUMMARY Coordinating angiogenesis with acquisition of tissue-specific properties in endothelial cells is essential for vascular function. In the retina, endothelial cells form a blood-retina barrier by virtue of tight junctions and low transcytosis. While the canonical Norrin/Fz4/Lrp5/6 pathway is essential for angiogenesis, vascular remodeling, and barrier maturation, how these diverse processes are coordinated remains poorly understood. Here we demonstrate that Apcdd1, a negative regulator of Wnt/β-catenin signaling, is expressed in retinal endothelial cells during angiogenesis and barrier formation. Apcdd1-deficient mice exhibit a transient increase in vessel density at ages P10–P12 due to delayed vessel pruning. Moreover, Apcdd1 mutant endothelial cells precociously form the paracellular component of the barrier. Conversely, mice that overexpress Apcdd1 in retina endothelial cells have reduced vessel density but increased paracellular barrier permeability. Apcdd1 thus serves to precisely modulate Wnt/Norrin signaling activity in the retinal endothelium and coordinate the timing of both vascular pruning and barrier maturation.
Neurovascular unit and barrier maturation rely on vascular basement membrane (vBM) composition. Laminins, a major vBM component, are crucial for these processes, yet the signaling pathway(s) that regulate their expression remain unknown. Here, we show that mural cells have active Wnt/β-catenin signaling during central nervous system development in mice. Bulk RNA sequencing and validation using postnatal day 10 and 14 wild-type versus adenomatosis polyposis coli downregulated 1 (Apcdd1−/−) mouse retinas revealed that Lama2 mRNA and protein levels are increased in mutant vasculature with higher Wnt/β-catenin signaling. Mural cells are the main source of Lama2, and Wnt/β-catenin activation induces Lama2 expression in mural cells in vitro. Markers of mature astrocytes, including aquaporin 4 (a water channel in astrocyte endfeet) and integrin-α6 (a laminin receptor), are upregulated in Apcdd1−/− retinas with higher Lama2 vBM deposition. Thus, the Wnt/β-catenin pathway regulates Lama2 expression in mural cells to promote neurovascular unit and barrier maturation.
Mucolipidosis IV (MLIV) is an ultra-rare, recessively inherited lysosomal disorder resulting from inactivating mutations in MCOLN1, the gene encoding the lysosomal cation channel TRPML1. The disease primarily affects the central nervous system (CNS) and manifests in the first year with cognitive and motor developmental delay, followed by a gradual decline in neurological function across the second decade of life, blindness, and premature death in third or fourth decades. Brain pathology manifestations in MLIV are consistent with hypomyelinating leukodystrophy with brain iron accumulation. Presently, there are no approved or investigational therapies for MLIV, and pathogenic mechanisms remain largely unknown. The MLIV mouse model, Mcoln1−/− mice, recapitulates all major manifestations of the human disease. Here, to better understand the pathological mechanisms in the MLIV brain, we performed cell type specific LC–MS/MS proteomics analysis in the MLIV mouse model and reconstituted molecular signatures of the disease in either freshly isolated populations of neurons, astrocytes, oligodendrocytes, and neural stem cells, or whole tissue cortical homogenates from young adult symptomatic Mcoln1−/− mice. Our analysis confirmed on the molecular level major histopathological hallmarks of MLIV universally present in Mcoln1−/− tissue and brain cells, such as hypomyelination, lysosomal dysregulation, and impaired metabolism of lipids and polysaccharides. Importantly, pathway analysis in brain cells revealed mitochondria-related alterations in all Mcoln1−/− brain cells, except oligodendrocytes, that was not possible to resolve in whole tissue. We also report unique proteome signatures and dysregulated pathways for each brain cell population used in this study. These data shed new light on cell-intrinsic mechanisms of MLIV and provide new insights for biomarker discovery and validation to advance translational studies for this disease.
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