Autophagy is a highly conserved mechanism for cells to degrade dysfunctional organelles and damaged proteins via the lysosome. The normal function of autophagy-lysosomal pathway (ALP) is essential for maintaining the homeostasis for cells, especially the non-dividing neurons. Impairment of autophagy has been reported in neurodegenerative diseases (NDs), such as Alzheimer's disease (AD) and Parkinson's disease (PD), which are generally aging-related disorders characterized by aberrant accumulation of pathological protein aggregates. Therefore, enhancing autophagic clearance of these deposits in patient brain becomes a potential therapeutic strategy for the NDs. Transcription factor EB (TFEB) is the key regulator of autophagy function and lysosomal biogenesis by initiating the transcription of related genes in the nucleus. Chemical or genetic activation of TFEB showed abilities to ameliorate the beta amyloid (Aß) and tau pathologies, which are two dominant molecular hallmarks of AD, in various AD models. Congruently, pharmaceutical enhancement of TFEB function also exerted hopeful neuroprotection in PD models through improving the clearance of pathological factors including mutant a-synuclein (a-syn) deposits and damaged mitochondria to enhance neuron survival. Given the importance of TFEB in regulating autophagy-lysosomal pathway and the therapeutical potential of TFEB for NDs, we harnessed a series of screening to identify novel chemical and genetic regulators of TFEB. Firstly, a high-content screening (HCS) was conducted to seek the compounds that could increase the T FEB nuclear translocation from a library of natural products isolated from traditional Chinese medicine (TCM). Betulinaldehyde (Betu), a compound belongs to the pentacyclic triterpenoid, could robustly increase the shuttling of TFEB from the cytoplasm to nucleus. Betu increased the formation of autolysosomes and improved lysosomal biogenesis indicating the activation of autophagy-lysosomal pathway, in an mTOR-independent manner. We subsequently performed the second round HCS screening with a compound library consisting of kinase inhibitors to search the molecular target of Betu. Specifically inhibiting Janus kinase 3 (JAK3) almost totally blocked Betu-mediated TFEB nuclear translocation. In addition, we performed a mass spectrometry (MS) analysis and identified several serine residues, including S 109, S114, S122, S142, and S211, of TFEB were dephosphorylated. Intriguingly, Betu-induced TFEB activation was inhibited by FK506 and cyclosporin A, the inhibitors of serine/threonine protein phosphatase calcineurin. In light of these observations, we proposed that Betu activated TFEB in JAK3 and calcineurin-dependent manner. To investigate the neuroprotective effects of Betu, we recruited in vitro and in vivo PD models. Betu increased the autophagic clearance of cellular u-syn in PC12 cells overexpressing the A53T mutant a-syn proteins. In line with the in vitro data, Betu reduced the accumulation of pathological a-syn in the midbrain of the transgenic mice expressing the A53T mutant a-syn proteins and ameliorated their motor ability tested in the CatWalk gait analysis. To identify novel genetic regulators of TFEB, a genome-scale CRISPR/Cas9 knockout (GeCKO) screening was harnessed in HeLa cells stably expressing 3xFlag-TFEB and lysophosphatidic acid receptor 1 (LPARI), a G protein-coupled receptor (GPCR) locates on the cell membrane, was identified as a negative regulator of TFEB nuclear translocation through the screening. Chemically or genetically inhibiting LPARI leaded to TFEB-mediated activation of autophagy-lysosomal pathway. To evaluate the therapeutic potential of LPARI inhibitor in the Alzheimer disease model, ONO-7300243 (ONO), the specific inhibitor of LPARI, were tested in cellular and mice AD models. ONO exhibited neuroprotective capacity in improving the autophagic clearance of Aß and p-Tau in AD cell models and ameliorated the memory loss in 3xTg AD mice. To sum up, a robust activator (Betu) of TFEB was identified from the TCM-extracted compound library and a negative regulatory gene (LPARI) of TFEB was identified through the GeCKO screening. Our study provided more alternatives to target TFEB-mediated ALP activation and highlighted the therapeutic potential of autophagic clearance in these aberrant protein aggregates-related neurodegenerative disorders.