Chaperone-mediated autophagy sustains haematopoietic stem-cell function.

The activation of mostly quiescent haematopoietic stem cells (HSCs) is a prerequisite for life-long production of blood cells1. This process requires major molecular adaptations to allow HSCs to meet the regulatory and metabolic requirements for cell division2-4. The mechanisms that govern cellular reprograming upon stem-cell activation, and the subsequent return of stem cells to quiescence, have not been fully characterized. Here we show that chaperone-mediated autophagy (CMA)5, a selective form of lysosomal protein degradation, is involved in sustaining HSC function in adult mice. CMA is required for protein quality control in stem cells and for the upregulation of fatty acid metabolism upon HSC activation. We find that CMA activity in HSCs decreases with age and show that genetic or pharmacological activation of CMA can restore the functionality of old mouse and human HSCs. Together, our findings provide mechanistic insights into a role for CMA in sustaining quality control, appropriate energetics and overall long-term HSC function. Our work suggests that CMA may be a promising therapeutic target for enhancing HSC function in conditions such as ageing or stem-cell transplantation.

Exosomes from Hair Follicle Epidermal Neural Crest Stem Cells Promote Acellular Nerve Allografts to Bridge Rat Facial Nerve Defects.

Previous studies showed that acellular nerve allografts (ANAs) have been successfully utilized in repairing peripheral nerve defects, and exosomes produced by stem cells are useful in supporting axon regrowth after peripheral nerve injury. In this study, exosomes from hair follicle epidermal neural crest stem cells (EPI-NCSCs-Exos) combined with ANAs were used to bridge facial nerve defects. EPI-NCSCs-Exos were isolated by ultracentrifuge, and were identified. After coculture, EPI-NCSCs-Exos were internalized into dorsal root ganglions (DRGs) and schwann cells (SCs) in vitro, respectively. EPI-NCSCs-Exos elongate the length of axons and dendrites of DRGs, and accelerated the proliferation and migration of SCs, and increased neurotrophic factor expression of SCs as well. The next step was to assign 24 Sprague Dawley male rats randomly and equally into three groups: the autograft group, the ANA group, and the ANA + EPI-NCSCs-Exos group. Each rat manufactured a 5-mm gap of facial nerve defect and immediately bridged by the corresponding transplants, respectively. After surgery, behavioral changes and electrophysiological testing of each rat were observed and assessed. At 90 days postoperatively, the retrogradely fluorescent tracer-labeled neurons were successfully observed on the injured side in the three groups. Morphological changes of facial nerve regeneration were evaluated by transmission electron microscopy and semithin toluidine blue staining. The results showed that nerve fiber density, nerve fiber diameter, and myelin sheath thickness in the ANA group were significantly worse than those in the other two groups (P < 0.05). No significant difference in nerve fiber density and myelin sheath thickness was observed between the autograft group and the ANA + EPI-NCSCs-Exos group (P = 0.14; P = 0.23). Our data indicated that EPI-NCSCs-Exos facilitate ANAs to bridge facial nerve defects and have the potential to replace autograft therapy in clinic.

Chaperone-mediated autophagy regulates adipocyte differentiation.

Adipogenesis is a tightly orchestrated multistep process wherein preadipocytes differentiate into adipocytes. The most studied aspect of adipogenesis is its transcriptional regulation through timely expression and silencing of a vast number of genes. However, whether turnover of key regulatory proteins per se controls adipogenesis remains largely understudied. Chaperone-mediated autophagy (CMA) is a selective form of lysosomal protein degradation that, in response to diverse cues, remodels the proteome for regulatory purposes. We report here the activation of CMA during adipocyte differentiation and show that CMA regulates adipogenesis at different steps through timely degradation of key regulatory signaling proteins and transcription factors that dictate proliferation, energetic adaptation, and signaling changes required for adipogenesis.

Targeting retinoic acid receptor alpha-corepressor interaction activates chaperone-mediated autophagy and protects against retinal degeneration.

Chaperone-mediated autophagy activity, essential in the cellular defense against proteotoxicity, declines with age, and preventing this decline in experimental genetic models has proven beneficial. Here, we have identified the mechanism of action of selective chaperone-mediated autophagy activators previously developed by our group and have leveraged that information to generate orally bioavailable chaperone-mediated autophagy activators with favorable brain exposure. Chaperone-mediated autophagy activating molecules stabilize the interaction between retinoic acid receptor alpha - a known endogenous inhibitor of chaperone-mediated autophagy - and its co-repressor, nuclear receptor corepressor 1, resulting in changes of a discrete subset of the retinoic acid receptor alpha transcriptional program that leads to selective chaperone-mediated autophagy activation. Chaperone-mediated autophagy activators molecules activate this pathway in vivo and ameliorate retinal degeneration in a retinitis pigmentosa mouse model. Our findings reveal a mechanism for pharmacological targeting of chaperone-mediated autophagy activation and suggest a therapeutic strategy for retinal degeneration.

MAEA is an E3 ubiquitin ligase promoting autophagy and maintenance of haematopoietic stem cells.

Haematopoietic stem cells (HSCs) tightly regulate their quiescence, proliferation, and differentiation to generate blood cells during the entire lifetime. The mechanisms by which these critical activities are balanced are still unclear. Here, we report that Macrophage-Erythroblast Attacher (MAEA, also known as EMP), a receptor thus far only identified in erythroblastic island, is a membrane-associated E3 ubiquitin ligase subunit essential for HSC maintenance and lymphoid potential. Maea is highly expressed in HSCs and its deletion in mice severely impairs HSC quiescence and leads to a lethal myeloproliferative syndrome. Mechanistically, we have found that the surface expression of several haematopoietic cytokine receptors (e.g. MPL, FLT3) is stabilised in the absence of Maea, thereby prolonging their intracellular signalling. This is associated with impaired autophagy flux in HSCs but not in mature haematopoietic cells. Administration of receptor kinase inhibitor or autophagy-inducing compounds rescues the functional defects of Maea-deficient HSCs. Our results suggest that MAEA provides E3 ubiquitin ligase activity, guarding HSC function by restricting cytokine receptor signalling via autophagy.

Regulation of energy homeostasis by the ubiquitin-independent REGγ proteasome.

Maintenance of energy homeostasis is essential for cell survival. Here, we report that the ATP- and ubiquitin-independent REGγ-proteasome system plays a role in maintaining energy homeostasis and cell survival during energy starvation via repressing rDNA transcription, a major intracellular energy-consuming process. Mechanistically, REGγ-proteasome limits cellular rDNA transcription and energy consumption by targeting the rDNA transcription activator SirT7 for ubiquitin-independent degradation under normal conditions. Moreover, energy starvation induces an AMPK-directed SirT7 phosphorylation and subsequent REGγ-dependent SirT7 subcellular redistribution and degradation, thereby further reducing rDNA transcription to save energy to overcome cell death. Energy starvation is a promising strategy for cancer therapy. Our report also shows that REGγ knockdown markedly improves the anti-tumour activity of energy metabolism inhibitors in mice. Our results underscore a control mechanism for an ubiquitin-independent process in maintaining energy homeostasis and cell viability under starvation conditions, suggesting that REGγ-proteasome inhibition has a potential to provide tumour-starving benefits.

A TiO2-Au-polymer hybrid system for the photoelectrochemical immunoassay of SirT1.

Human sirtuin1 (SirT1), which is a member of the sirtuin family, plays an important role in a wide range of cellular processes. Here we demonstrate a new strategy for the photoelectrochemical assay of SirT1 in different cell lines based on a semiconductor-polymer hybrid system consisting of Au-polymer and TiO2-Au nanocomposites. Au-polymer (GC-HBAP) hybrids were synthesized from crosslinked hyperbranched azo-polymer and gold colloids and then used as an immobilization platform for SirT1 antibody. Gold-doped TiO2 (TiO2-Au) nanocomposites were prepared as the photoelectrochemical labels for signal readout in the sandwiched immunoassay. The integration of GC-HBAP with TiO2-Au facilitated the electron transfer and the photoelectrocatalytic reaction, resulting in good analytical performance with high sensitivity, selectivity and rapid response for the analysis of SirT1 levels in different cell lines. This proposed semiconductor-polymer system might open a new perspective for the development of a highly sensitive photoelectrochemical immunosensor, and have potentially promising applications in assays of other proteins.

[Sero-epidemiological investigation on hepatitis B among permanent residents in Shenzhen area].

OBJECTIVE: To understand the infection status and epidemiological features of HBV in permanent residents of Shenzhen city. METHODS: A multi-stage stratified random sampling method was performed for questionnaire survey to permanently-registered residents of 1-59 years old in Luohu and Baoan district of Shenzhen in 2010, and blood samples of the subjects were collected. Hepatitis B virus-related surface antigen (HBsAg) and hepatitis B virus surface antibody (anti-HBs) were detected with ELISA. RESULTS: The total 3771 studied population showed 252 HBsAg positive and 2712 anti-HBs positive residents with the standardization prevalence as 9.73% and 72.83% , respectively. The difference of the prevalence of HBsAg and anti-HBs between males and females were not statistically significant (P > 0.05). The prevalence of HBsAg was reduced with increasing age. The differences of the prevalence of HBsAg between Shenzhen permanent registered and non-permanent registered population were not significant, but the prevalence of anti-HBs in Permanent registered residents (78.32%) was higher than in non-permanent (66.03%, χ(2) = 41.613, P < 0.001). The prevalence of HBsAg was significantly different in various occupational and educational levels. Peasants had the highest prevalence (24.13%) and medical workers had the highest prevalence of anti-HBs (89.10% ). People with junior high school education had the highest prevalence of HBsAg (12.76%) and the lowest of anti-HBs (62.45%). Population with high-level education had the highest prevalence of anti-HBs(81.00% average). The prevalence of HBsAg was over 10% in people who were born in Shenzhen and Guangdong province, and the anti-HBs was the highest in Shenzhen population with the prevalence as 74.48% and 76.47% , respectively. CONCLUSION: In the Shenzhen resident population, the overall prevalence of HBV was lower than the average level of Guangdong province, but higher than the national wide.

The REGγ proteasome regulates hepatic lipid metabolism through inhibition of autophagy.

The ubiquitin-proteasome and autophagy-lysosome systems are major proteolytic pathways, whereas function of the Ub-independent proteasome pathway is yet to be clarified. Here, we investigated roles of the Ub-independent REGγ-proteasome proteolytic system in regulating metabolism. We demonstrate that mice deficient for the proteasome activator REGγ exhibit dramatic autophagy induction and are protected against high-fat diet (HFD)-induced liver steatosis through autophagy. Molecularly, prevention of steatosis in the absence of REGγ entails elevated SirT1, a deacetylase regulating autophagy and metabolism. REGγ physically binds to SirT1, promotes its Ub-independent degradation, and inhibits its activity to deacetylate autophagy-related proteins, thereby inhibiting autophagy under normal conditions. Moreover, REGγ and SirT1 dissociate from each other through a phosphorylation-dependent mechanism under energy-deprived conditions, unleashing SirT1 to stimulate autophagy. These observations provide a function of the REGγ proteasome in autophagy and hepatosteatosis, underscoring mechanistically a crosstalk between the proteasome and autophagy degradation system in the regulation of lipid homeostasis.