Most nervous system disorders manifest through alterations in neuronal signaling based on abnormalities in neuronal excitability, synaptic transmission, and cell survival. However, such neuronal phenotypes are frequently accompanied or even caused by metabolic dysfunctions in neuronal or nonneuronal cells. The tight packing and highly heterogenous properties of neural, glial and vascular cell types pose significant challenges to dissecting metabolic aspects of brain disorders. Perturbed cholesterol homeostasis has recently emerged as key parameter associated with sub-sets of neurodevelopmental disorders. However, approaches for tracking and visualizing endogenous cholesterol distribution in the brain have limited capability of resolving cell type-specific differences. We here develop tools for genetically-encoded sensors that report on cholesterol distribution in the mouse brain with cellular resolution. We apply these probes to examine sub-cellular cholesterol accumulation in two genetic mouse models of neurodevelopmental disorders, Npc1 and Ptchd1 knock-out mice. While both genes encode proteins with sterol-sensing domains that have been implicated in cholesterol transport, we uncover highly selective and cell type-specific phenotypes in cholesterol homeostasis. The tools established in this work should facilitate probing sub-cellular cholesterol distribution in complex tissues like the mammalian brain and enable capturing cell type-specific alterations in cholesterol flow between cells in models of brain disorders.
Synaptic vesicle proteins (SVps) are thought to travel in heterogeneous carriers dependent on the motor UNC-104/KIF1A. In C. elegans neurons, we found that some SVps are transported along with lysosomal proteins by the motor UNC-104/KIF1A. LRK-1/LRRK2 and the clathrin adaptor protein complex AP-3 are critical for the separation of lysosomal proteins from SVp transport carriers. In lrk-1 mutants, both SVp carriers and SVp carriers containing lysosomal proteins are independent of UNC-104, suggesting that LRK-1 plays a key role in ensuring UNC-104-dependent transport of SVps. Additionally, LRK-1 likely acts upstream of the AP-3 complex and regulates the membrane localization of AP-3. The action of AP-3 is necessary for the active zone protein SYD-2/Liprin-α to facilitate the transport of SVp carriers. In the absence of the AP-3 complex, SYD-2/Liprin-α acts with UNC-104 to instead facilitate the transport of SVp carriers containing lysosomal proteins. We further show that the mistrafficking of SVps into the dendrite in lrk-1 and apb-3 mutants depends on SYD-2, likely by regulating the recruitment of the AP-1/UNC-101. We propose that SYD-2 acts in concert with both the AP-1 and AP-3 complexes to ensure polarized trafficking of SVps.
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