Linking lysosomal trafficking defects with changes in aging and stress response in Drosophila

Defects in pathways that direct cellular components to the lysosome for degradation are often linked with a decrease in viability and with progressive disorders. Previously we had shown that blue cheese (bchs: Drosophila homologue of human Alfy) mutations lead to reduced longevity and the accumulation of ubiquitinated neural aggregates. A genetic modifier screen based on overexpression of Bchs in the eye was used to identify several potential genetic interactions, which included autophagic and endocytic trafficking genes as well as cytoskeletal and motor proteins and members of the SUMO and ubiquitin signaling pathways. We found that mutations in several of the genes identified in the screen also result in bchs-like phenotypes, including a reduction in adult lifespan and changes in ubiquitinated protein profiles. In addition, we show here that Bchs modifiers belonging to the autophagic and trans-Golgi trafficking pathways also display defects in adult starvation response. Our data further support a role for Bchs/Alfy in the autophagic pathway and strongly indicate that autophagy plays an important role in aging and stress response.     

Genetic modifiers of the Drosophila blue cheese gene link defects in lysosomal transport with decreased life span and altered ubiquitinated-protein profiles

Defects in lysosomal trafficking pathways lead to decreased cell viability and are associated with progressive disorders in humans. Previously we have found that loss-of-function (LOF) mutations in the Drosophila gene blue cheese (bchs) lead to reduced adult life span, increased neuronal death, and widespread CNS degeneration that is associated with the formation of ubiquitinated-protein aggregates. To identify potential genes that participate in the bchs functional pathway, we conducted a genetic modifier screen based on alterations of an eye phenotype that arises from high-level overexpression of Bchs. We found that mutations in select autophagic and endocytic trafficking genes, defects in cytoskeletal and motor proteins, as well as mutations in the SUMO and ubiquitin signaling pathways behave as modifiers of the Bchs gain-of-function (GOF) eye phenotype. Individual mutant alleles that produced viable adults were further examined for bchs-like phenotypes. Mutations in several lysosomal trafficking genes resulted in significantly decreased adult life spans and several mutants showed changes in ubiquitinated protein profiles as young adults. This work represents a novel approach to examine the role that lysosomal transport and function have on adult viability. The genes characterized in this study have direct human homologs, suggesting that similar defects in lysosomal transport may play a role in human health and age-related processes.     

Ubiquitin Trafficking to the Lysosome: Keeping the House Tidy and Getting Rid of Unwanted Guests

Bacterial killing by autophagic delivery to the lysosomal compartment has been shown for Mycobacteria, Streptococcus, Shigella, Legionella and Salmonella, indicating an important role for this conserved trafficking pathway for the control of intracellular bacterial pathogens. In a recent study we found that solubilized lysosomes isolated from bone marrow-derived macrophages had potent antibacterial properties against M. tuberculosis and M. smegmatis that were associated with ubiquitin and ubiquitin-derived peptides. We propose that ubiquitinated proteins are delivered to the lysosomal compartment, where degradation by lysosomal proteinases generates ubiquitin-derived peptides with antimycobacterial properties. This surprising finding provokes a number of questions regarding the nature and trafficking of ubiquitin and ubiquitin-modified proteins in mammalian cells. We discuss the possible role(s) that the multivesicular body (MVB), the late endosome and the autophagosome may play in trafficking of ubiquitinated proteins to the lysosome.

Addendum to:

Lysosomal Killing of Mycobacterium Mediated by Ubiquitin-Derived Peptides is Enhanced by Autophagy

S. Alonso, K. Pethe, D.G. Russell and G.E. Purdy

Proc Natl Acad Sci USA 2007; 104:6031-6     

A genome‐wide screen identifies genes that suppress the accumulation of spontaneous mutations in young and aged yeast cells

To ensure proper transmission of genetic information, cells need to preserve and faithfully replicate their genome, and failure to do so leads to genome instability, a hallmark of both cancer and aging. Defects in genes involved in guarding genome stability cause several human progeroid syndromes, and an age‐dependent accumulation of mutations has been observed in different organisms, from yeast to mammals. However, it is unclear whether the spontaneous mutation rate changes during aging and whether specific pathways are important for genome maintenance in old cells. We developed a high‐throughput replica‐pinning approach to screen for genes important to suppress the accumulation of spontaneous mutations during yeast replicative aging. We found 13 known mutation suppression genes, and 31 genes that had no previous link to spontaneous mutagenesis, and all acted independently of age. Importantly, we identified PEX19, encoding an evolutionarily conserved peroxisome biogenesis factor, as an age‐specific mutation suppression gene. While wild‐type and pex19Δ young cells have similar spontaneous mutation rates, aged cells lacking PEX19 display an elevated mutation rate. This finding suggests that functional peroxisomes may be important to preserve genome integrity specifically in old cells.     

Functional multivesicular bodies are required for autophagic clearance of protein aggregates associated with neurodegenerative disease

The endosomal sorting complexes required for transport (ESCRTs) are required to sort integral membrane proteins into intralumenal vesicles of the multivesicular body (MVB). Mutations in the ESCRT-III subunit CHMP2B were recently associated with frontotemporal dementia and amyotrophic lateral sclerosis (ALS), neurodegenerative diseases characterized by abnormal ubiquitin-positive protein deposits in affected neurons. We show here that autophagic degradation is inhibited in cells depleted of ESCRT subunits and in cells expressing CHMP2B mutants, leading to accumulation of protein aggregates containing ubiquitinated proteins, p62 and Alfy. Moreover, we find that functional MVBs are required for clearance of TDP-43 (identified as the major ubiquitinated protein in ALS and frontotemporal lobar degeneration with ubiquitin deposits), and of expanded polyglutamine aggregates associated with Huntington's disease. Together, our data indicate that efficient autophagic degradation requires functional MVBs and provide a possible explanation to the observed neurodegenerative phenotype seen in patients with CHMP2B mutations.     

Alfy-dependent elimination of aggregated proteins by macroautophagy: Can there be too much of a good thing?

Degradation of different cargo by macroautophagy is emerging as a highly selective process which relies upon specific autophagy receptors and adapter molecules that link the cargo with the autophagic molecular machinery. We have recently reported that the large phsophatidylinositol-3-phosphate (PtdIns(3)P)-binding protein Alfy (Autophagy-linked FYVE protein) is required for selective degradation of aggregated proteins. Although depletion of Alfy inhibits Atg5-dependent aggregate degradation, overexpression of Alfy results in Atg5-dependent aggregate clearance and neuroprotection. Alfy-mediated degradation requires the ability of Alfy to directly interact with Atg5. This ability to interact with the core autophagic machinery may cause Alfy to diminish the responsiveness to nonselective autophagic degradation as measured by long-lived protein degradation. Thus, increasing Alfy-mediated protein degradation may be beneficial in some organs, but may be detrimental in others.Key words: autophagy, protein aggregates, neurodegeneration, Alfy, aggregation, degradation     

ESCRTing autophagic clearance of aggregating proteins

Autophagy has recently been found to play an important role in the degradation of damaged macromolecules, in particular misfolded, aberrant proteins that can disrupt neuronal function and cause neurodegeneration if not removed. Mutations in the Endosomal Sorting Complex Required for Transport (ESCRT)-III subunit CHMP2B were recently associated with frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), neurodegenerative diseases characterized by abnormal ubiquitin-positive protein deposits in affected neurons. The ESCRT proteins are known to sort ubiquitinated integral membrane proteins into intralumenal vesicles of the multivesicular body (MVB), but it was not known how ESCRT mutations could cause neurodegenerative disease. We found autophagic degradation to be inhibited in cells depleted of ESCRT subunits or expressing CHMP2B mutants and in Drosphila melanogaster lacking ESCRTs. In addition to accumulation of autophagic vesicles, we also found increased levels of membrane-free ubiquitin-positive protein aggregates in ESCRT-depleted cells. Using cellular and Drosophila models for Huntington’s disease, we showed that reduced ESCRT levels inhibit clearance of expanded polyglutamine aggregates and aggravate their neurotoxic effect. Together, our data indicate that efficient autophagic degradation requires functional MVBs and provide a possible explanation to the observed neurodegenerative phenotype seen in patients with CHMP2B mutations. In this addendum we discuss models to explain the functions of ESCRTs and MVBs in autophagic degradation.

Addendum to: Filimonenko M, Stuffers S, Raiborg C, Yamamoto A, Malerod L, Fisher EM, Isaacs A, Brech A, Stenmark H, Simonsen A. Functional multivesicular bodies are required for autophagic clearance of protein aggregates associated with neurodegenerative disease. J Cell Biol 2007; 179;485-500.

and

Rusten TE, Vaccari T, Lindmo K, Rodahl LM, Nezis IP, Sem-Jacobsen C, Wendler F, Vincent JP, Brech A, Bilder D, Stenmark H. ESCRTs and Fab1 regulate distinct steps of autophagy. Curr Biol 2007; 17;1817-25.     

Promoting basal levels of autophagy in the nervous system enhances longevity and oxidant resistance in adult Drosophila

Autophagy is involved with the turnover of intracellular components and the management of stress responses. Genetic studies in mice have shown that suppression of neuronal autophagy can lead to the accumulation of protein aggregates and neurodegeneration. However, no study has shown that increasing autophagic gene expression can be beneficial to an aging nervous system. Here we demonstrate that expression of several autophagy genes is reduced in Drosophila neural tissues as a normal part of aging. The age-dependent suppression of autophagy occurs concomitantly with the accumulation of insoluble ubiquitinated proteins (IUP), a marker of neuronal aging and degeneration. Mutations in the Atg8a gene (autophagy-related 8a) result in reduced lifespan, IUP accumulation and increased sensitivity to oxidative stress. In contrast, enhanced Atg8a expression in older fly brains extends the average adult lifespan by 56% and promotes resistance to oxidative stress and the accumulation of ubiquitinated and oxidized proteins. These data indicate that genetic or age-dependent suppression of autophagy is closely associated with the buildup of cellular damage in neurons and a reduced lifespan, while maintaining the expression of a rate-limiting autophagy gene prevents the age-dependent accumulation of damage in neurons and promotes longevity.     

A Genetic Screen in Drosophila Reveals Novel Cytoprotective Functions of the Autophagy-Lysosome Pathway

The highly conserved autophagy-lysosome pathway is the primary mechanism for breakdown and recycling of macromolecular and organellar cargo in the eukaryotic cell. Autophagy has recently been implicated in protection against cancer, neurodegeneration, and infection, and interest is increasing in additional roles of autophagy in human health, disease, and aging. To search for novel cytoprotective features of this pathway, we carried out a genetic mosaic screen for mutations causing increased lysosomal and/or autophagic activity in the Drosophila melanogaster larval fat body. By combining Drosophila genetics with live-cell imaging of the fluorescent dye LysoTracker Red and fixed-cell imaging of autophagy-specific fluorescent protein markers, the screen was designed to identify essential metazoan genes whose disruption causes increased flux through the autophagy-lysosome pathway. The screen identified a large number of genes associated with the protein synthesis and ER-secretory pathways (e.g. aminoacyl tRNA synthetases, Oligosaccharyl transferase, Sec61α), and with mitochondrial function and dynamics (e.g. Rieske iron-sulfur protein, Dynamin-related protein 1). We also observed that increased lysosomal and autophagic activity were consistently associated with decreased cell size. Our work demonstrates that disruption of the synthesis, transport, folding, or glycosylation of ER-targeted proteins at any of multiple steps leads to autophagy induction. In addition to illuminating cytoprotective features of autophagy in response to cellular damage, this screen establishes a genetic methodology for investigating cell biological phenotypes in live cells, in the context of viable wild type organisms.     

Insulin htts on Autophagy

Macroautophagocytosis has been shown to participate in the degradation and clearance of polyglutamine (polyQ) tract-containing proteins generated by trinucleotide repeat expansion mutations. Large expansions are the genetic cause of diseases such as Huntington’s disease that lead to neuronal dysfunction due to polyQ protein aggregates. Recently, a functional screen performed by Yamamoto et al. to investigate proteins that regulate such autophagic processes revealed a novel role for insulin signaling in the promotion of autophagy of mutant protein aggregates. This suggests that insulin/insulin-like growth factor signaling pathways not only prevent the induction of autophagy, but paradoxically may promote autophagy of deleterious proteins in certain circumstances.

Commentary to:

Autophagy-Mediated Clearance of Huntingtin Aggregates Triggered by the Insulin-Signaling Pathway

A. Yamamoto, M.L. Cremona and J.E. Rothman

J Cell Biol 2006; 172:719-31     

Mutations affecting pigmentation and shape of the adult zebrafish

 Mutations causing a visible phenotype in the adult serve as valuable visible genetic markers in multicellular genetic model organisms such as Drosophila melanogaster, Caenorhabditis elegans and Arabidopsis thaliana. In a large scale screen for mutations affecting early development of the zebrafish, we identified a number of mutations that are homozygous viable or semiviable. Here we describe viable mutations which produce visible phenotypes in the adult fish. These predominantly affect the fins and pigmentation, but also the eyes and body length of the adult. A number of dominant mutations caused visible phenotypes in the adult fish. Mutations in three genes, long fin, another long fin and wanda affected fin formation in the adult. Four mutations were found to cause a dominant reduction of the overall body length in the adult. The adult pigment pattern was found to be changed by dominant mutations in wanda, asterix, obelix, leopard, salz and pfeffer. Among the recessive mutations producing visible phenotypes in the homozygous adult, a group of mutations that failed to produce melanin was assayed for tyrosinase activity. Mutations in sandy produced embryos that failed to express tyrosinase activity. These are potentially useful for using tyrosinase as a marker for the generation of transgenic lines of zebrafish. Received: 17 June 1996 / Accepted: 15 July 1996     

Drosophila Gyf/GRB10 interacting GYF protein is an autophagy regulator that controls neuron and muscle homeostasis

Autophagy is an essential process for eliminating ubiquitinated protein aggregates and dysfunctional organelles. Defective autophagy is associated with various degenerative diseases such as Parkinson disease. Through a genetic screening in Drosophila, we identified CG11148, whose product is orthologous to GIGYF1 (GRB10-interacting GYF protein 1) and GIGYF2 in mammals, as a new autophagy regulator; we hereafter refer to this gene as Gyf. Silencing of Gyf completely suppressed the effect of Atg1-Atg13 activation in stimulating autophagic flux and inducing autophagic eye degeneration. Although Gyf silencing did not affect Atg1-induced Atg13 phosphorylation or Atg6-Pi3K59F (class III PtdIns3K)-dependent Fyve puncta formation, it inhibited formation of Atg13 puncta, suggesting that Gyf controls autophagy through regulating subcellular localization of the Atg1-Atg13 complex. Gyf silencing also inhibited Atg1-Atg13-induced formation of Atg9 puncta, which is accumulated upon active membrane trafficking into autophagosomes. Gyf-null mutants also exhibited substantial defects in developmental or starvation-induced accumulation of autophagosomes and autolysosomes in the larval fat body. Furthermore, heads and thoraxes from Gyf-null adults exhibited strongly reduced expression of autophagosome-associated Atg8a-II compared to wild-type (WT) tissues. The decrease in Atg8a-II was directly correlated with an increased accumulation of ubiquitinated proteins and dysfunctional mitochondria in neuron and muscle, which together led to severe locomotor defects and early mortality. These results suggest that Gyf-mediated autophagy regulation is important for maintaining neuromuscular homeostasis and preventing degenerative pathologies of the tissues. Since human mutations in the GIGYF2 locus were reported to be associated with a type of familial Parkinson disease, the homeostatic role of Gyf-family proteins is likely to be evolutionarily conserved.     

Disruption of Drosophila melanogaster Lipid Metabolism Genes Causes Tissue Overgrowth Associated with Altered Developmental Signaling

Developmental patterning requires the precise interplay of numerous intercellular signaling pathways to ensure that cells are properly specified during tissue formation and organogenesis. The spatiotemporal function of many developmental pathways is strongly influenced by the biosynthesis and intracellular trafficking of signaling components. Receptors and ligands must be trafficked to the cell surface where they interact, and their subsequent endocytic internalization and endosomal trafficking is critical for both signal propagation and its down-modulation. In a forward genetic screen for mutations that alter intracellular Notch receptor trafficking in Drosophila melanogaster, we recovered mutants that disrupt genes encoding serine palmitoyltransferase and acetyl-CoA carboxylase. Both mutants cause Notch, Wingless, the Epidermal Growth Factor Receptor (EFGR), and Patched to accumulate abnormally in endosomal compartments. In mosaic animals, mutant tissues exhibit an unusual non-cell-autonomous effect whereby mutant cells are functionally rescued by secreted activities emanating from adjacent wildtype tissue. Strikingly, both mutants display prominent tissue overgrowth phenotypes that are partially attributable to altered Notch and Wnt signaling. Our analysis of the mutants demonstrates genetic links between abnormal lipid metabolism, perturbations in developmental signaling, and aberrant cell proliferation.     

Alfy-dependent elimination of aggregated proteins by macroautophagy

Degradation of different cargo by macroautophagy is emerging as a highly selective process which relies upon specific autophagy receptors and adapter molecules that link the cargo with the autophagic molecular machinery. We have recently reported that the large phsophatidylinositol-3-phosphate (PtdIns(3)P)-binding protein Alfy (Autophagy-linked FYVE protein) is required for selective degradation of aggregated proteins. Although depletion of Alfy inhibits Atg5-dependent aggregate degradation, overexpression of Alfy results in Atg5-dependent aggregate clearance and neuroprotection. Alfy-mediated degradation requires the ability of Alfy to directly interact with Atg5. This ability to interact with the core autophagic machinery may cause Alfy to diminish the responsiveness to non-selective autophagic degradation as measured by long-lived protein degradation. Thus, increasing Alfy-mediated protein degradation may be beneficial in some organs, but may be detrimental in others.     

Pyramid Screening: Combining Three Genetic Screens into One Efficient Screen for Shoot Regeneration Mutants in Arabidopsis thaliana

Gain-of-function genetic mutants are typically found by creating a non-permissive condition and screening for plants that overcome the stress. Separate genetic screens are conducted for each condition, a potentially time-consuming effort. In severed Arabidopsis thaliana leaves, high light, suboptimal hormone exposure and old age were each independently found to reduce frequency of shoot regeneration. Rather than conducting three separate mutant screens to dissect these three pathways, a laborious process, we hypothesized that we could undertake a single economical screen to retrieve mutations specific for each trait as well as cross-talk alleles between pathways. Instead of creating non-permissive conditions for each of our three traits of interest, we combined the three suboptimal stress conditions such that only when combined was shoot regeneration abolished. No one stress was primarily responsible for loss of our trait, thus ensuring that we could recover mutant alleles in any of the three pathways of interest. Screening of 18,000 mutagenized plants resulted in 12 SHOOTING UP (stu) mutants. Secondary screening revealed that we had recovered alleles that were both specific for a pathway (light, hormones or age) and which acted through multiple pathways. Our approach, which we refer to as pyramid screening, represents an economical method for mutant screening of multiple pathways in parallel (three screens in one) and has the potential to recover alleles that cross-talk between multiple pathways that underlie a complex trait such as organ regeneration. Pyramid screening should be widely applicable across species.

     

Detection of Mutations in Tumor Suppressor Genes

Defects in vital genes occur in a high percentage of human diseases, including cancer. Defects could be due to the accumulation of mutations in the genes leading to the production of faulty proteins. Although the biological significance of such mutant proteins still remains in question, recent experiments have demonstrated that genes overproducing faulty proteins are often associated with tumor cell growth. Thep53tumor suppressor gene is the most frequently mutated gene yet identified in human cancer. It is mutated in wide variety of human cancers. Missense mutations are common for thep53gene and are essential for the transforming ability of the oncogene. The wild-typep53gene may directly suppress cell growth or indirectly activate genes that are involved in growth suppression. Thus inactivation of wild-typep53by point mutation may contribute to transformation. Therefore, identification of such mutations have potential clinical implications. Recently, polymerase chain reaction-based advanced molecular techniques had a profound impact on the detection and identification of such mutations. These techniques are sensitive and quantitative tools for the study of the pathogenesis of neoplastic diseases at the single-cell level.     

Functional annotation of mouse mutations in embryonic stem cells by use of expression profiling

Expression profiling offers a potential high-throughput phenotype screen for mutant mouse embryonic stem (ES) cells. We have assessed the ability of expression arrays to distinguish among heterozygous mutant ES cell lines and to accurately reflect the normal function of the mutated genes. Two ES cell lines hemizygous for overlapping regions of mouse Chromosome (Chr) 5 differed substantially from the wildtype parental line and from each other. Expression differences included frequent downregulation of hemizygous genes and downstream effects on genes mapping to other chromosomes. Some genes were affected similarly in each deletion line, consistent with the overlap of the deletions. To determine whether such downstream effects reveal pathways impacted by a mutation, we examined ES cell lines heterozygous for mutations in either of two well-characterized genes. A heterozygous mutation in the gene encoding the cell cycle regulator, cyclin D kinase 4 (Cdk4), affected expression of many genes involved in cell growth and proliferation. A heterozygous mutation in the ATP binding cassette transporter family A, member 1 (Abca1) gene, altered genes associated with lipid homeostasis, the cytoskeleton, and vesicle trafficking. Heterozygous Abca1 mutation had similar effects in liver, indicating that ES cell expression profile reflects changes in fundamental processes relevant to mutant gene function in multiple cell types.