Harnessing chaperone-mediated autophagy for the degradation of endogenous proteins

Rapid and reversible methods for altering the function of endogenous proteins are not only indispensable tools for probing complex biological systems, but may potentially drive the development of new therapeutics for the treatment of human diseases. Genetic approaches have provided insights into protein function, but are limited in speed, reversibility and spatiotemporal control. To overcome these limitations, we have developed a peptide-based method to degrade a given endogenous protein at the post-translational level by harnessing chaperone-mediated autophagy, a major intracellular protein degradation pathway mediated by the lysosome. This thesis presents the design and validation of SNIPER (Selective Native Protein ERadication) for use in cultured cells as well as in intact animals. Specifically, we demonstrate the specificity, efficacy and generalizability of SNIPER by showing efficient knockdown of various proteins, including death-associated protein kinase 1 (160 kDa), scaffolding protein PSD-95 (95 kDa) and α-synuclein (19 kDa), with their respective SNIPER peptides in cell lines and rat neuronal cultures. Moreover, we examine the characteristics of SNIPER-mediated protein knockdown and found that the level of protein knockdown is dose-, time- and lysosome-dependent. Furthermore, we demonstrate that SNIPER efficiently knocked down a dephosphorylated subpopulation of a given protein while sparing its phosphorylated form, attesting to the specificity of the method. Finally, using a rat model of focal ischemic stroke, we show that a single intravenous injection of a SNIPER peptide efficiently knocked down a death-promoting protein in the brains of freely moving rats and protected the rat brain from ischemic injury. Taken together, SNIPER is a robust and convenient research tool for manipulating the levels of endogenous proteins in situ, and may also lead to the development of novel protein knockdown–based therapeutics for treating human diseases.Medicine, Faculty ofGraduat

Age-related loss of neural stem cell O-GlcNAc promotes a glial fate switch through STAT3 activation

Increased neural stem cell (NSC) quiescence is a major determinant of age-related regenerative decline in the adult hippocampus. However, a coextensive model has been proposed in which division-coupled conversion of NSCs into differentiated astrocytes restrict the stem cell pool with age. Here we report that age-related loss of the posttranslational modification, O-linked β-N-acetylglucosamine (O-GlcNAc), in NSCs promotes a glial fate switch. We detect an age-dependent decrease in NSC O-GlcNAc levels coincident with decreased neurogenesis and increased gliogenesis in the mature hippocampus. Mimicking an age-related loss of NSC O-GlcNAcylation in young mice reduces neurogenesis, increases astrocyte differentiation, and impairs associated cognitive function. Using RNA-sequencing of primary NSCs following decreased O-GlcNAcylation, we detected changes in the STAT3 signaling pathway indicative of glial differentiation. Moreover, using O-GlcNAc-specific mass spectrometry analysis of the aging hippocampus, together with an in vitro site-directed mutagenesis approach, we identify loss of STAT3 O-GlcNAc at Threonine 717 as a driver of astrocyte differentiation. Our data identify the posttranslational modification, O-GlcNAc, as a key molecular regulator of regenerative decline underlying an age-related NSC fate switch

One-pot hydrodeoxygenation of biomass furan derivatives into decane under mild conditions over Pd/C combined with phosphotungstic acid

One-pot hydrodeoxygenation (HDO) of biomass furan derivatives into long-chain alkanes remains unclear owing to the complicated reactions at high temperatures. Here, Pd/C combined with phosphotungstic acid (HPW), a strong Bronsted solid acid promoter, was developed to produce decane under mild conditions through the one-pot HDO of delta-furfurylidenelevulinic acid (FDLA). A 93.16% sum yield of alkanes including 89.53% yield of decane was obtained under 3 MPa H-2 at 170 degrees C for 4 h. The ring-opening and HDO of furans are accomplished at preheating stage, which enable to clarify the complicated reaction pathway. In addition, experimental validations and catalytic characterizations confirm that the synergistic effect of active acid-metal sites plays a critical role in the generation of alkanes via the formation and hydrogenolysis of lactone intermediates to aldehyde/alcohol. This work provides guidance for the one-pot HDO of biomass furan derivatives via the combination of metal catalyst and strong Bronsted solid acid under mild conditions

Blood factors transfer beneficial effects of exercise on neurogenesis and cognition to the aged brain

Reversing brain aging may be possible through systemic interventions such as exercise. We found that administration of circulating blood factors in plasma from exercised aged mice transferred the effects of exercise on adult neurogenesis and cognition to sedentary aged mice. Plasma concentrations of glycosylphosphatidylinositol (GPI)-specific phospholipase D1 (Gpld1), a GPI-degrading enzyme derived from liver, were found to increase after exercise and to correlate with improved cognitive function in aged mice, and concentrations of Gpld1 in blood were increased in active, healthy elderly humans. Increasing systemic concentrations of Gpld1 in aged mice ameliorated age-related regenerative and cognitive impairments by altering signaling cascades downstream of GPI-anchored substrate cleavage. We thus identify a liver-to-brain axis by which blood factors can transfer the benefits of exercise in old age