Jung R, Lechler MC, Fernandez-Villegas A, Chung CW, Jones HC, Choi YH, Thompson MA, Rödelsperger C, Röseler W, Kaminski Schierle GS, Sommer RJ, David DC Signalling

During aging, proteostasis capacity declines and distinct proteins become unstable and can accumulate as protein aggregates inside and outside of cells. Both in disease and during aging, proteins selectively aggregate in certain tissues and not others. Yet, tissue-specific regulation of cytoplasmic protein aggregation remains poorly understood. Surprisingly, we found that the inhibition of 3 core protein quality control systems, namely chaperones, the proteasome, and macroautophagy, leads to lower levels of age-dependent protein aggregation in Caenorhabditis elegans pharyngeal muscles, but higher levels in body-wall muscles. We describe a novel safety mechanism that selectively targets newly synthesized proteins to suppress their aggregation and associated proteotoxicity. The safety mechanism relies on macroautophagy-independent lysosomal degradation and involves several previously uncharacterized components of the intracellular pathogen response (IPR). We propose that this protective mechanism engages an anti-aggregation machinery targeting aggregating proteins for lysosomal degradation.

+view abstract PLoS biology, PMID: 37708127 Sep 2023

Gallotta I, Sandhu A, Peters M, Haslbeck M, Jung R, Agilkaya S, Blersch JL, Rödelsperger C, Röseler W, Huang C, Sommer RJ, David DC Signalling

In metazoans, the secreted proteome participates in intercellular signalling and innate immunity, and builds the extracellular matrix scaffold around cells. Compared with the relatively constant intracellular environment, conditions for proteins in the extracellular space are harsher, and low concentrations of ATP prevent the activity of intracellular components of the protein quality-control machinery. Until now, only a few bona fide extracellular chaperones and proteases have been shown to limit the aggregation of extracellular proteins. Here we performed a systematic analysis of the extracellular proteostasis network in Caenorhabditis elegans with an RNA interference screen that targets genes that encode the secreted proteome. We discovered 57 regulators of extracellular protein aggregation, including several proteins related to innate immunity. Because intracellular proteostasis is upregulated in response to pathogens, we investigated whether pathogens also stimulate extracellular proteostasis. Using a pore-forming toxin to mimic a pathogenic attack, we found that C. elegans responded by increasing the expression of components of extracellular proteostasis and by limiting aggregation of extracellular proteins. The activation of extracellular proteostasis was dependent on stress-activated MAP kinase signalling. Notably, the overexpression of components of extracellular proteostasis delayed ageing and rendered worms resistant to intoxication. We propose that enhanced extracellular proteostasis contributes to systemic host defence by maintaining a functional secreted proteome and avoiding proteotoxicity.

+view abstract Nature, PMID: 32641833 08 2020

Huang C, Wagner-Valladolid S, Stephens AD, Jung R, Poudel C, Sinnige T, Lechler MC, Schlörit N, Lu M, Laine RF, Michel CH, Vendruscolo M, Kaminski CF, Kaminski Schierle GS, David DC Signalling

Reduced protein homeostasis leading to increased protein instability is a common molecular feature of aging, but it remains unclear whether this is a cause or consequence of the aging process. In neurodegenerative diseases and other amyloidoses, specific proteins self-assemble into amyloid fibrils and accumulate as pathological aggregates in different tissues. More recently, widespread protein aggregation has been described during normal aging. Until now, an extensive characterization of the nature of age-dependent protein aggregation has been lacking. Here, we show that age-dependent aggregates are rapidly formed by newly synthesized proteins and have an amyloid-like structure resembling that of protein aggregates observed in disease. We then demonstrate that age-dependent protein aggregation accelerates the functional decline of different tissues in . Together, these findings imply that amyloid-like aggregates contribute to the aging process and therefore could be important targets for strategies designed to maintain physiological functions in the late stages of life.

+view abstract eLife, PMID: 31050339 03 05 2019

Groh N, Gallotta I, Lechler MC, Huang C, Jung R, David DC Signalling

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional.

+view abstract Journal of visualized experiments : JoVE, PMID: 29286457 26 11 2017

Lechler MC, David DC Signalling

Low complexity (LC) prion-like domains are over-represented among RNA-binding proteins (RBPs) and contribute to the dynamic nature of RNA granules. Importantly, several neurodegenerative diseases are characterized by cytoplasmic "solid" aggregates formed by mainly nuclear RBPs harboring LC prion-like domains. Although RBP aggregation in disease has been extensively characterized, it remains unknown how the process of aging disturbs RBP dynamics. Our recent study revealed that RNA granule components including 2 key stress granule RBPs with LC prion-like domains, PAB-1 and TIAR-2, aggregate in aged Caenorhabditis elegans in the absence of disease. Here we present new evidence showing that sustained stress granule formation triggers RBP aggregation. In addition, we demonstrate that mild chronic stress during aging promotes mislocalization of nuclear RBPs. We discuss the consequences of aberrant interactions between age-related RBP aggregation and disease-associated RBP aggregation. In particular, we show that FUST-1 and PAB-1 co-localize in aberrant cytoplasmic accumulations. Significantly, long-lived animals with reduced insulin/IGF-1 signaling abrogate stress granule RBP aggregation through activation of the transcription factors HSF-1 and DAF-16. We evaluate the different mechanisms that could maintain dynamic stress granules. Together these findings highlight how changes with age could contribute to pathogenesis in neurodegenerative diseases and disruption of RNA homeostasis.

+view abstract Prion, PMID: 28956717 03 Sep 2017

Groh N, Bühler A, Huang C, Li KW, van Nierop P, Smit AB, Fändrich M, Baumann F, David DC Signalling

Aging is the most important risk factor for neurodegenerative diseases associated with pathological protein aggregation such as Alzheimer's disease. Although aging is an important player, it remains unknown which molecular changes are relevant for disease initiation. Recently, it has become apparent that widespread protein aggregation is a common feature of aging. Indeed, several studies demonstrate that 100s of proteins become highly insoluble with age, in the absence of obvious disease processes. Yet it remains unclear how these misfolded proteins aggregating with age affect neurodegenerative diseases. Importantly, several of these aggregation-prone proteins are found as minor components in disease-associated hallmark aggregates such as amyloid-β plaques or neurofibrillary tangles. This co-localization raises the possibility that age-dependent protein aggregation directly contributes to pathological aggregation. Here, we show for the first time that highly insoluble proteins from aged or aged mouse brains, but not from young individuals, can initiate amyloid-β aggregation . We tested the seeding potential at four different ages across the adult lifespan of . Significantly, protein aggregates formed during the early stages of aging did not act as seeds for amyloid-β aggregation. Instead, we found that changes in protein aggregation occurring during middle-age initiated amyloid-β aggregation. Mass spectrometry analysis revealed several late-aggregating proteins that were previously identified as minor components of amyloid-β plaques and neurofibrillary tangles such as 14-3-3, Ubiquitin-like modifier-activating enzyme 1 and Lamin A/C, highlighting these as strong candidates for cross-seeding. Overall, we demonstrate that widespread protein misfolding and aggregation with age could be critical for the initiation of pathogenesis, and thus should be targeted by therapeutic strategies to alleviate neurodegenerative diseases.

+view abstract Frontiers in aging neuroscience, PMID: 28567012 2017

Jansen IE, Ye H, Heetveld S, Lechler MC, Michels H, Seinstra RI, Lubbe SJ, Drouet V, Lesage S, Majounie E, Gibbs JR, Nalls MA, Ryten M, Botia JA, Vandrovcova J, Simon-Sanchez J, Castillo-Lizardo M, Rizzu P, Blauwendraat C, Chouhan AK, Li Y, Yogi P, Amin N, van Duijn CM, , Morris HR, Brice A, Singleton AB, David DC, Nollen EA, Jain S, Shulman JM, Heutink P Signalling

Whole-exome sequencing (WES) has been successful in identifying genes that cause familial Parkinson's disease (PD). However, until now this approach has not been deployed to study large cohorts of unrelated participants. To discover rare PD susceptibility variants, we performed WES in 1148 unrelated cases and 503 control participants. Candidate genes were subsequently validated for functions relevant to PD based on parallel RNA-interference (RNAi) screens in human cell culture and Drosophila and C. elegans models.

+view abstract Genome biology, PMID: 28137300 30 01 2017

Lechler MC, Crawford ED, Groh N, Widmaier K, Jung R, Kirstein J, Trinidad JC, Burlingame AL, David DC Signalling

Low-complexity "prion-like" domains in key RNA-binding proteins (RBPs) mediate the reversible assembly of RNA granules. Individual RBPs harboring these domains have been linked to specific neurodegenerative diseases. Although their aggregation in neurodegeneration has been extensively characterized, it remains unknown how the process of aging disturbs RBP dynamics. We show that a wide variety of RNA granule components, including stress granule proteins, become highly insoluble with age in C. elegans and that reduced insulin/insulin-like growth factor 1 (IGF-1) daf-2 receptor signaling efficiently prevents their aggregation. Importantly, stress-granule-related RBP aggregates are associated with reduced fitness. We show that heat shock transcription factor 1 (HSF-1) is a main regulator of stress-granule-related RBP aggregation in both young and aged animals. During aging, increasing DAF-16 activity restores dynamic stress-granule-related RBPs, partly by decreasing the buildup of other misfolded proteins that seed RBP aggregation. Longevity-associated mechanisms found to maintain dynamic RBPs during aging could be relevant for neurodegenerative diseases.

+view abstract Cell reports, PMID: 28076789 10 01 2017

David DC Signalling

For all organisms promoting protein homeostasis is a high priority in order to optimize cellular functions and resources. However, there is accumulating evidence that aging leads to a collapse in protein homeostasis and widespread non-disease protein aggregation. This review examines these findings and discusses the potential causes and consequences of this physiological aggregation with age in particular in relation to disease protein aggregation and toxicity. Importantly, recent evidence points to unexpected differences in protein-quality-control and susceptibility to protein aggregation between neurons and other cell types. In addition, new insight into the cell-non-autonomous coordination of protein homeostasis by neurons will be presented.

+view abstract Frontiers in genetics, PMID: 23181070 2012

Crawford ED, Seaman JE, Barber AE, David DC, Babbitt PC, Burlingame AL, Wells JA Signalling

Caspases, cysteine proteases with aspartate specificity, are key players in programmed cell death across the metazoan lineage. Hundreds of apoptotic caspase substrates have been identified in human cells. Some have been extensively characterized, revealing key functional nodes for apoptosis signaling and important drug targets in cancer. But the functional significance of most cuts remains mysterious. We set out to better understand the importance of caspase cleavage specificity in apoptosis by asking which cleavage events are conserved across metazoan model species. Using N-terminal labeling followed by mass spectrometry, we identified 257 caspase cleavage sites in mouse, 130 in Drosophila, and 50 in Caenorhabditis elegans. The large majority of the caspase cut sites identified in mouse proteins were found conserved in human orthologs. However, while many of the same proteins targeted in the more distantly related species were cleaved in human orthologs, the exact sites were often different. Furthermore, similar functional pathways are targeted by caspases in all four species. Our data suggest a model for the evolution of apoptotic caspase specificity that highlights the hierarchical importance of functional pathways over specific proteins, and proteins over their specific cleavage site motifs.

+view abstract Cell death and differentiation, PMID: 22918439 Dec 2012

David DC, Ollikainen N, Trinidad JC, Cary MP, Burlingame AL, Kenyon C Signalling

Aberrant protein aggregation is a hallmark of many age-related diseases, yet little is known about whether proteins aggregate with age in a non-disease setting. Using a systematic proteomics approach, we identified several hundred proteins that become more insoluble with age in the multicellular organism Caenorhabditis elegans. These proteins are predicted to be significantly enriched in beta-sheets, which promote disease protein aggregation. Strikingly, these insoluble proteins are highly over-represented in aggregates found in human neurodegeneration. We examined several of these proteins in vivo and confirmed their propensity to aggregate with age. Different proteins aggregated in different tissues and cellular compartments. Protein insolubility and aggregation were significantly delayed or even halted by reduced insulin/IGF-1-signaling, which also slows aging. We found a significant overlap between proteins that become insoluble and proteins that influence lifespan and/or polyglutamine-repeat aggregation. Moreover, overexpressing one aggregating protein enhanced polyglutamine-repeat pathology. Together our findings indicate that widespread protein insolubility and aggregation is an inherent part of aging and that it may influence both lifespan and neurodegenerative disease.

+view abstract PLoS biology, PMID: 20711477 10 Aug 2010

David DC, Ittner LM, Gehrig P, Nergenau D, Shepherd C, Halliday G, Götz J Signalling

Alzheimer's disease (AD) is characterized by Abeta peptide-containing plaques and tau-containing neurofibrillary tangles (NFTs). Both pathologies have been combined by crossing Abeta plaque-forming APP mutant mice with NFT-forming P301L tau mutant mice or by stereotaxically injecting beta-amyloid peptide 1-42 (Abeta42) into brains of P301L tau mutant mice. In cell culture, Abeta42 induces filamentous tau aggregates. To understand which processes are disrupted by Abeta42 in the presence of tau aggregates, we applied comparative proteomics to Abeta42-treated P301L tau-expressing neuroblastoma cells and the amygdala of P301L tau transgenic mice stereotaxically injected with Abeta42. Remarkably, a significant fraction of proteins altered in both systems belonged to the same functional categories, i.e. stress response and metabolism. We also identified model-specific effects of Abeta42 treatment such as differences in cell signaling proteins in the cellular model and of cytoskeletal and synapse associated proteins in the amygdala. By Western blotting (WB) and immunohistochemistry (IHC), we were able to show that 72% of the tested candidates were altered in human AD brain with a major emphasis on stress-related unfolded protein responsive candidates. These data highlight these processes as potentially important initiators in the Abeta42-mediated pathogenic cascade in AD and further support the role of unfolded proteins in the course of AD.

+view abstract Proteomics, PMID: 17111439 Dec 2006

David DC, Hoerndli F, Götz J Signalling

Transcriptomics and proteomics are increasingly applied to gain a mechanistic insight into neurodegenerative disorders. These techniques not only identify distinct, differentially expressed mRNAs and proteins but are also employed to dissect signaling pathways and reveal networks by using an integrated approach. In part I of this back-to-back review, technical aspects are discussed: in the transcriptomics section, which includes enrichment by laser microcapture dissection, we comment on qRT-PCR, SAGE, subtractive hybridization, differential display and microarrays, including software packages. In the proteomics section we discuss two-dimensional (2D) gel electrophoresis, liquid chromatography, methods to label and enrich specific proteins or peptides, and different types of mass spectrometers. These tools have been applied to a range of neurodegenerative disorders and are discussed and integrated in part II (Functional Genomics meets neurodegenerative disorders. Part II: application and data integration).

+view abstract Progress in neurobiology, PMID: 16168556 Jun 2005

David DC, Hauptmann S, Scherping I, Schuessel K, Keil U, Rizzu P, Ravid R, Dröse S, Brandt U, Müller WE, Eckert A, Götz J Signalling

Transgenic mice overexpressing the P301L mutant human tau protein exhibit an accumulation of hyperphosphorylated tau and develop neurofibrillary tangles. The consequences of tau pathology were investigated here by proteomics followed by functional analysis. Mainly metabolism-related proteins including mitochondrial respiratory chain complex components, antioxidant enzymes, and synaptic proteins were identified as modified in the proteome pattern of P301L tau mice. Significantly, the reduction in mitochondrial complex V levels in the P301L tau mice revealed using proteomics was also confirmed as decreased in human P301L FTDP-17 (frontotemporal dementia with parkinsonism linked to chromosome 17) brains. Functional analysis demonstrated a mitochondrial dysfunction in P301L tau mice together with reduced NADH-ubiquinone oxidoreductase activity and, with age, impaired mitochondrial respiration and ATP synthesis. Mitochondrial dys-function was associated with higher levels of reactive oxygen species in aged transgenic mice. Increased tau pathology as in aged homozygous P301L tau mice revealed modified lipid peroxidation levels and the up-regulation of antioxidant enzymes in response to oxidative stress. Furthermore, P301L tau mitochondria displayed increased vulnerability toward beta-amyloid (Abeta) peptide insult, suggesting a synergistic action of tau and Abeta pathology on the mitochondria. Taken together, we conclude that tau pathology involves a mitochondrial and oxidative stress disorder possibly distinct from that caused by Abeta.

+view abstract The Journal of biological chemistry, PMID: 15831501 24 Jun 2005

David DC, Layfield R, Serpell L, Narain Y, Goedert M, Spillantini MG Signalling

Filamentous inclusions composed of the microtubule-associated protein tau are a defining characteristic of a large number of neurodegenerative diseases. Here we show that tau degradation in stably transfected and non-transfected SH-SY5Y cells is blocked by the irreversible proteasome inhibitor lactacystin. Further, we find that in vitro, natively unfolded tau can be directly processed by the 20S proteasome without a requirement for ubiquitylation, and that a highly reproducible pattern of degradation intermediates is readily detectable during this process. Analysis of these intermediates shows that 20S proteasomal processing of tau is bi-directional, proceeding from both N- and C-termini, and that populations of relatively stable intermediates arise probably because of less efficient digestion of the C-terminal repeat region. Our results are consistent with an in vivo role for the proteasome in tau degradation and support the existence of ubiquitin-independent pathways for the proteasomal degradation of unfolded proteins.

+view abstract Journal of neurochemistry, PMID: 12358741 Oct 2002