Upregulation of autophagy may have therapeutic benefit in a range of diseases that include neurodegenerative conditions caused by intracytosolic aggregate-prone proteins, such as Huntington's ...disease, and certain infectious diseases, such as tuberculosis. The best-characterized drug that enhances autophagy is rapamycin, an inhibitor of the TOR (target of rapamycin) proteins, which are widely conserved from yeast to man. Unfortunately, the side effects of rapamycin, especially immunosuppression, preclude its use in treating certain diseases including tuberculosis, which accounts for approximately 2 million deaths worldwide each year, spurring interest in finding novel drugs that selectively enhance autophagy. We have recently reported a novel two-step screening process for the discovery of such compounds. We first identified compounds that enhance the growth-inhibitory effects of rapamycin in the budding yeast Saccharomyces cerevisiae, which we termed small molecule enhancers of rapamycin (SMERs). Next we showed that three SMERs induced autophagy independently, or downstream of mTOR, in mammalian cells, and furthermore enhanced the clearance of a mutant huntingtin fragment in cell disease models. These SMERs also protected against mutant huntingtin fragment toxicity in Drosophila. We have subsequently tested two of the SMERs in models of tuberculosis and both enhance the killing of mycobacteria by primary human macrophages.
Addendum to:
Small Molecules Enhance Autophagy and Reduce Toxicity in Huntington's Disease Models
S. Sarkar, E.O. Perlstein, S. Imarisio, S. Pineau, A. Cordenier, R.L. Maglathlin, J.A. Webster, T.A. Lewis, C.J. O'Kane, S.L. Schreiber and D.C. Rubinsztein
Nat Chem Biol 2007; 3:331-8
Autophagy is an intracellular degradation pathway for malfunctioning aggregation-prone proteins, damaged organelles, unwanted macromolecules and invading pathogens. This process is essential for ...maintaining cellular and tissue homeostasis that contribute to organismal survival. Autophagy dysfunction has been implicated in the pathogenesis of diverse human diseases, and therefore, therapeutic exploitation of autophagy is of potential biomedical relevance. A number of chemical screening approaches have been established for the drug discovery of autophagy modulators based on the perturbations of autophagy reporters or the clearance of autophagy substrates. These readouts can be detected by fluorescence and high-content microscopy, flow cytometry, microplate reader and immunoblotting, and the assays have evolved to enable high-throughput screening and measurement of autophagic flux. Several pharmacological modulators of autophagy have been identified that act either via the classical mechanistic target of rapamycin (mTOR) pathway or independently of mTOR. Many of these autophagy modulators have been demonstrated to exert beneficial effects in transgenic models of neurodegenerative disorders, cancer, infectious diseases, liver diseases, myopathies as well as in lifespan extension. This review describes the commonly used chemical screening approaches in mammalian cells and the key autophagy modulators identified through these methods, and highlights the therapeutic benefits of these compounds in specific disease contexts.
Autophagy, a catabolic process governing cellular and energy homeostasis, is essential for cell survival and human health. Here, we present a protocol for generating autophagy-deficient (ATG5−/−) ...human neurons from human embryonic stem cell (hESC)-derived neural precursors. We describe steps for analyzing loss of autophagy by immunoblotting. We then detail analysis of cell death by luminescence-based cytotoxicity assay and fluorescence-based TUNEL staining. This hESC-based experimental platform provides a genetic knockout model for undertaking autophagy studies relevant to human biology.
For complete details on the use and execution of this protocol, please refer to Sun et al. (2023).1
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•Protocol for differentiation of hESC-derived neural precursors into neurons•Characterization of autophagy deficiency through immunoblot analysis of LC3 and p62•Analysis of cell death in hESC-derived neurons by cytotoxicity and TUNEL assays•Loss of autophagy causes cell death in ATG5−/− hESC-derived neurons
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
Autophagy, a catabolic process governing cellular and energy homeostasis, is essential for cell survival and human health. Here, we present a protocol for generating autophagy-deficient (ATG5−/−) human neurons from human embryonic stem cell (hESC)-derived neural precursors. We describe steps for analyzing loss of autophagy by immunoblotting. We then detail analysis of cell death by luminescence-based cytotoxicity assay and fluorescence-based TUNEL staining. This hESC-based experimental platform provides a genetic knockout model for undertaking autophagy studies relevant to human biology.
Overexpression of transcription factors has been used to directly reprogram somatic cells into a range of other differentiated cell types, including multipotent neural stem cells (NSCs), that can be ...used to generate neurons and glia. However, the ability to maintain the NSC state independent of the inducing factors and the identity of the somatic donor cells remain two important unresolved issues in transdifferentiation. Here we used transduction of doxycycline-inducible transcription factors to generate stable tripotent NSCs. The induced NSCs (iNSCs) maintained their characteristics in the absence of exogenous factor expression and were transcriptionally, epigenetically, and functionally similar to primary brain-derived NSCs. Importantly, we also generated tripotent iNSCs from multiple adult cell types, including mature liver and B cells. Our results show that self-maintaining proliferative neural cells can be induced from nonectodermal cells by expressing specific combinations of transcription factors.
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•Transgene-independent tripotent neural stem cells are induced from somatic cells•H3K27ac enhancer profiling shows iNSCs are epigenetically reprogrammed genome-wide•Adult liver and B cells are lineage converted to iNSCs using a “secondary” system•iNSCs derived from B lymphocytes have immuloglobulin loci rearrangements
In this article, Jaenisch and colleagues generate induced neural stem cells that are transcriptionally, epigenetically, and functionally similar to primary NSCs by transiently overexpressing transcription factors in fibroblasts. The authors then demonstrate lineage conversion by inducing tripotent iNSCs from multiple adult cell types, including liver cells and B cells with genetic rearrangements.
Oculopharyngeal muscular dystrophy (OPMD) is caused by a trinucleotide repeat expansion mutation in the coding region of the gene encoding PABPN1 (polyadenylate-binding protein nuclear 1). Mutant ...PABPN1 with a polyalanine tract expansion forms aggregates within the nuclei of skeletal muscle fibers. There is currently no effective treatment. We have developed cell and mouse models of OPMD and have identified the aggregation of mutant PABPN1 and apoptosis as therapeutic targets. Here, we show that transglutaminase activity is increased in muscle from OPMD model mice. Elevated transglutaminase 2 expression enhances, whereas TG2 knockdown suppresses, the toxicity and aggregation of mutant PABPN1 in cells. Cystamine protects against the toxicity of mutant PABPN1 and exerts its effect via the inhibition of transglutaminase 2, as cystamine treatment is unable to further reduce the protective effect of transglutaminase 2 knockdown on mutant PABPN1 toxicity in cells. Cystamine also reduces the aggregation and toxicity of mutant PABPN1 in human cells. In a mouse model of OPMD, cystamine treatment reduced the elevated transglutaminase activity, attenuated muscle weakness, reduced aggregate load, and decreased apoptotic markers in muscle. Therefore, inhibitors of transglutaminase 2 should be considered as possible therapeutics for OPMD.
Autophagy has been implicated in various physiological and disease conditions in recent years. A number of small molecule modulators have been identified, both as tools and as potential therapeutics. ...Despite extensive characterization of autophagy in yeast, mammalian autophagy pathways are not fully understood. Recently, calcium phosphate precipitates (CPP), which are used to transfect DNA into cells, were reported to induce autophagy, when assayed up to 6 h after treatment. Because of the widespread use of this reagent, we attempted to confirm these findings. Consistent with the previous study, we showed that CPP induces autophagosome synthesis at early time-points, such as 4 h and 6 h. However, at 24 h after treatment, we were surprised to see that autophagy flux was reduced, due to impaired autophagosome-lysosome fusion. At this time point, there was an accumulation of autophagy substrates and the formation of abnormally large autophagosomes. Thus, one may need to consider assaying autophagy modulators at different time-points with a range of assays in order to understand their effects. Finally, the complex consequences of CPP on autophagy suggest that it is best avoided as a transfection reagent in studies aiming to analyse autophagy itself, or processes that are modulated by autophagy, like apoptosis.
DNA double-strand breaks (DSBs) can cause chromosomal rearrangements and extensive loss of heterozygosity (LOH), hallmarks of cancer cells. Yet, how such events are normally suppressed is unclear. ...Here we identify roles for the DNA damage checkpoint pathway in facilitating homologous recombination (HR) repair and suppressing extensive LOH and chromosomal rearrangements in response to a DSB. Accordingly, deletion of Rad3ATR, Rad26ATRIP, Crb253BP1 or Cdc25 overexpression leads to reduced HR and increased break-induced chromosome loss and rearrangements. We find the DNA damage checkpoint pathway facilitates HR, in part, by promoting break-induced Cdt2-dependent nucleotide synthesis. We also identify additional roles for Rad17, the 9-1-1 complex and Chk1 activation in facilitating break-induced extensive resection and chromosome loss, thereby suppressing extensive LOH. Loss of Rad17 or the 9-1-1 complex results in a striking increase in break-induced isochromosome formation and very low levels of chromosome loss, suggesting the 9-1-1 complex acts as a nuclease processivity factor to facilitate extensive resection. Further, our data suggest redundant roles for Rad3ATR and Exo1 in facilitating extensive resection. We propose that the DNA damage checkpoint pathway coordinates resection and nucleotide synthesis, thereby promoting efficient HR repair and genome stability.
Fanconi anemia (FA) is a devastating genetic disease, associated with genomic instability and defects in DNA interstrand cross-link (ICL) repair. The FA repair pathway is not thought to be conserved ...in budding yeast, and although the yeast Mph1 helicase is a putative homolog of human FANCM, yeast cells disrupted for MPH1 are not sensitive to ICLs. Here, we reveal a key role for Mph1 in ICL repair when the Pso2 exonuclease is inactivated. We find that the yeast FANCM ortholog Mph1 physically and functionally interacts with Mgm101, a protein previously implicated in mitochondrial DNA repair, and the MutSα mismatch repair factor (Msh2-Msh6). Co-disruption of MPH1, MGM101, MSH6, or MSH2 with PSO2 produces a lesion-specific increase in ICL sensitivity, the elevation of ICL-induced chromosomal rearrangements, and persistence of ICL-associated DNA double-strand breaks. We find that Mph1-Mgm101-MutSα directs the ICL-induced recruitment of Exo1 to chromatin, and we propose that Exo1 is an alternative 5'-3' exonuclease utilised for ICL repair in the absence of Pso2. Moreover, ICL-induced Rad51 chromatin loading is delayed when both Pso2 and components of the Mph1-Mgm101-MutSα and Exo1 pathway are inactivated, demonstrating that the homologous recombination stages of ICL repair are inhibited. Finally, the FANCJ- and FANCP-related factors Chl1 and Slx4, respectively, are also components of the genetic pathway controlled by Mph1-Mgm101-MutSα. Together this suggests that a prototypical FA-related ICL repair pathway operates in budding yeast, which acts redundantly with the pathway controlled by Pso2, and is required for the targeting of Exo1 to chromatin to execute ICL repair.
Neurodegenerative diseases (NDDs) comprise conditions with impaired neuronal function and loss and may be associated with a build-up of aggregated proteins with altered physicochemical properties ...(misfolded proteins). There are many disorders, and causes include gene mutations, infections, or exposure to toxins. The autophagy pathway is involved in the removal of unwanted proteins and organelles through lysosomes. While lysosomal storage disorders have been described for many years, it is now recognised that perturbations of the autophagy pathway itself can also lead to neurodegenerative disease. These include monogenic disorders of key proteins involved in the autophagy pathway, and disorders within pathways that critically control autophagy through monitoring of the supply of nutrients (mTORC1 pathway) or of energy supply in cells (AMPK pathway). This review focuses on childhood-onset neurodegenerative disorders with perturbed autophagy, due to defects in the autophagy pathway, or in upstream signalling via mTORC1 and AMPK. The review first provides a short description of autophagy, as related to neurons. It then examines the extended role of autophagy in neuronal function, plasticity, and memory. There follows a description of each step of the autophagy pathway in greater detail, illustrated with examples of diseases grouped by the stage of their perturbation of the pathway. Each disease is accompanied by a short clinical description, to illustrate the diversity but also the overlap of symptoms caused by perturbation of key proteins necessary for the proper functioning of autophagy. Finally, there is a consideration of current challenges that need addressing for future therapeutic advances.
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•Autophagy is an important pathway for the removal of misfolded proteins from terminally differentiated neurons.•Monogenic defects in autophagy cause childhood-onset neurodegeneration.•Defects in different stages of the pathway may present with overlapping clinical features.•Increasing autophagic flux may be a therapeutic strategy to treat many autophagic disorders.
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