Towards a templated reaction that translates RNA in cells into a proaptotic peptide-PNA conjugate.

Nucleic acid-templated chemistry opens the intriguing prospect of triggering the synthesis of drugs only in diseased cells. Herein, we explore the feasibility of using RNA-templated chemical reactions for the activation of a known Smac peptidomimetic compound (SMC), which has proapoptotic activity. Two peptide nucleic acid (PNA) conjugates were used to enable conditional activation of a masked SMC by reduction of an azide either by Staudinger reduction or catalytic photoreduction using a ruthenium complex. The latter provided ~135 nM SMC-PNA on as little as 10 nM (0.01 eq.) template. For the evaluation of the templated azido-SMC reduction system in cellulo, a stable HEK 293 cell line was generated, which overexpressed a truncated, non-functional form of the XIAP mRNA target. We furthermore describe the development of electroporation protocols that enable a robust delivery of PNA conjugates into HEK 293 cells. The action of the reactive PNA conjugates was evaluated by viability and flow cytometric apoptosis assays. In addition, electroporated probes were re-isolated and analyzed by ultra-high performance liquid chromatography (UPLC). Unfortunately, the ruthenium-PNA conjugate proved phototoxic, and treatment of cells with PNA-linked reducing agent and the azido-masked SMC conjugate did not result in a greater viability loss than treatment with scrambled sequence controls. Intracellular product formation was not detectable. A control experiment in total cellular RNA isolate indicated that the templated reaction can in principle proceed in a complex system. The results of this first-of-its-kind study reveal the numerous hurdles that must be overcome if RNA molecules are to trigger the synthesis of pro-apoptotic drugs inside cells.

A conserved role for p48 homologs in protecting dopaminergic neurons from oxidative stress.

Parkinson's disease (PD) is the most common neurodegenerative movement disorder characterized by the progressive loss of dopaminergic (DA) neurons. Both environmental and genetic factors are thought to contribute to the pathogenesis of PD. Although several genes linked to rare familial PD have been identified, endogenous risk factors for sporadic PD, which account for the majority of PD cases, remain largely unknown. Genome-wide association studies have identified many single nucleotide polymorphisms associated with sporadic PD in neurodevelopmental genes including the transcription factor p48/ptf1a. Here we investigate whether p48 plays a role in the survival of DA neurons in Drosophila melanogaster and Caenorhabditis elegans. We show that a Drosophila p48 homolog, 48-related-2 (Fer2), is expressed in and required for the development and survival of DA neurons in the protocerebral anterior medial (PAM) cluster. Loss of Fer2 expression in adulthood causes progressive PAM neuron degeneration in aging flies along with mitochondrial dysfunction and elevated reactive oxygen species (ROS) production, leading to the progressive locomotor deficits. The oxidative stress challenge upregulates Fer2 expression and exacerbates the PAM neuron degeneration in Fer2 loss-of-function mutants. hlh-13, the worm homolog of p48, is also expressed in DA neurons. Unlike the fly counterpart, hlh-13 loss-of-function does not impair development or survival of DA neurons under normal growth conditions. Yet, similar to Fer2, hlh-13 expression is upregulated upon an acute oxidative challenge and is required for the survival of DA neurons under oxidative stress in adult worms. Taken together, our results indicate that p48 homologs share a role in protecting DA neurons from oxidative stress and degeneration, and suggest that loss-of-function of p48 homologs in flies and worms provides novel tools to study gene-environmental interactions affecting DA neuron survival.

Drosophila MIC10b can polymerize into cristae-shaping filaments.

Cristae are invaginations of the mitochondrial inner membrane that are crucial for cellular energy metabolism. The formation of cristae requires the presence of a protein complex known as MICOS, which is conserved across eukaryotic species. One of the subunits of this complex, MIC10, is a transmembrane protein that supports cristae formation by oligomerization. In Drosophila melanogaster, three MIC10-like proteins with different tissue-specific expression patterns exist. We demonstrate that CG41128/MINOS1b/DmMIC10b is the major MIC10 orthologue in flies. Its loss destabilizes MICOS, disturbs cristae architecture, and reduces the life span and fertility of flies. We show that DmMIC10b has a unique ability to polymerize into bundles of filaments, which can remodel mitochondrial crista membranes. The formation of these filaments relies on conserved glycine and cysteine residues, and can be suppressed by the co-expression of other Drosophila MICOS proteins. These findings provide new insights into the regulation of MICOS in flies, and suggest potential mechanisms for the maintenance of mitochondrial ultrastructure.

Maintenance of mitochondrial integrity in midbrain dopaminergic neurons governed by a conserved developmental transcription factor.

Progressive degeneration of dopaminergic (DA) neurons in the substantia nigra is a hallmark of Parkinson's disease (PD). Dysregulation of developmental transcription factors is implicated in dopaminergic neurodegeneration, but the underlying molecular mechanisms remain largely unknown. Drosophila Fer2 is a prime example of a developmental transcription factor required for the birth and maintenance of midbrain DA neurons. Using an approach combining ChIP-seq, RNA-seq, and genetic epistasis experiments with PD-linked genes, here we demonstrate that Fer2 controls a transcriptional network to maintain mitochondrial structure and function, and thus confers dopaminergic neuroprotection against genetic and oxidative insults. We further show that conditional ablation of Nato3, a mouse homolog of Fer2, in differentiated DA neurons causes mitochondrial abnormalities and locomotor impairments in aged mice. Our results reveal the essential and conserved role of Fer2 homologs in the mitochondrial maintenance of midbrain DA neurons, opening new perspectives for modeling and treating PD.

ATP hydrolysis by yeast Hsp104 determines protein aggregate dissolution and size in vivo.

Signs of proteostasis failure often entwine with those of metabolic stress at the cellular level. Here, we study protein sequestration during glucose deprivation-induced ATP decline in Saccharomyces cerevisiae. Using live-cell imaging, we find that sequestration of misfolded proteins and nascent polypeptides into two distinct compartments, stress granules, and Q-bodies, is triggered by the exhaustion of ATP. Both compartments readily dissolve in a PKA-dependent manner within minutes of glucose reintroduction and ATP level restoration. We identify the ATP hydrolase activity of Hsp104 disaggregase as the critical ATP-consuming process determining compartments abundance and size, even in optimal conditions. Sequestration of proteins into distinct compartments during acute metabolic stress and their retrieval during the recovery phase provide a competitive fitness advantage, likely promoting cell survival during stress.

Normal mitochondrial function in Saccharomyces cerevisiae has become dependent on inefficient splicing.

Self-splicing introns are mobile elements that have invaded a number of highly conserved genes in prokaryotic and organellar genomes. Here, we show that deletion of these selfish elements from the Saccharomyces cerevisiae mitochondrial genome is stressful to the host. A strain without mitochondrial introns displays hallmarks of the retrograde response, with altered mitochondrial morphology, gene expression and metabolism impacting growth and lifespan. Deletion of the complete suite of mitochondrial introns is phenocopied by overexpression of the splicing factor Mss116. We show that, in both cases, abnormally efficient transcript maturation results in excess levels of mature cob and cox1 host mRNA. Thus, inefficient splicing has become an integral part of normal mitochondrial gene expression. We propose that the persistence of S. cerevisiae self-splicing introns has been facilitated by an evolutionary lock-in event, where the host genome adapted to primordial invasion in a way that incidentally rendered subsequent intron loss deleterious.

Heat-induced longevity in budding yeast requires respiratory metabolism and glutathione recycling.

Heat-induced hormesis is a well-known conserved phenomenon in aging, traditionally attributed to the benefits conferred by increased amounts of heat shock (HS) proteins. Here we find that the key event for the HS-induced lifespan extension in budding yeast is the switch from glycolysis to respiratory metabolism. The resulting increase in reactive oxygen species activates the antioxidant response, supported by the redirection of glucose from glycolysis to the pentose phosphate pathway, increasing the production of NADPH. This sequence of events culminates in replicative lifespan (RLS) extension, implying decreased mortality per generation that persists even after the HS has finished. We found that switching to respiratory metabolism, and particularly the consequent increase in glutathione levels, were essential for the observed RLS extension. These results draw the focus away solely from the HS response and demonstrate that the antioxidant response has a key role in heat-induced hormesis. Our findings underscore the importance of the changes in cellular metabolic activity for heat-induced longevity in budding yeast.

TORC1-mediated sensing of chaperone activity alters glucose metabolism and extends lifespan.

Protein quality control mechanisms, required for normal cellular functioning, encompass multiple functions related to protein production and maintenance. However, the existence of communication between proteostasis and metabolic networks and its underlying mechanisms remain elusive. Here, we report that enhanced chaperone activity and consequent improved proteostasis are sensed by TORC1 via the activity of Hsp82. Chaperone enrichment decreases the level of Hsp82, which deactivates TORC1 and leads to activation of Snf1/AMPK, regardless of glucose availability. This mechanism culminates in the extension of yeast replicative lifespan (RLS) that is fully reliant on both TORC1 deactivation and Snf1/AMPK activation. Specifically, we identify oxygen consumption increase as the downstream effect of Snf1 activation responsible for the entire RLS extension. Our results set a novel paradigm for the role of proteostasis in aging: modulation of the misfolded protein level can affect cellular metabolic features as well as mitochondrial activity and consequently modify lifespan. The described mechanism is expected to open new avenues for research of aging and age-related diseases.

Crosstalk between cellular compartments protects against proteotoxicity and extends lifespan.

In cells living under optimal conditions, protein folding defects are usually prevented by the action of chaperones. Here, we investigate the cell-wide consequences of loss of chaperone function in cytosol, mitochondria or the endoplasmic reticulum (ER) in budding yeast. We find that the decline in chaperone activity in each compartment results in loss of respiration, demonstrating the dependence of mitochondrial activity on cell-wide proteostasis. Furthermore, each chaperone deficiency triggers a response, presumably via the communication among the folding environments of distinct cellular compartments, termed here the cross-organelle stress response (CORE). The proposed CORE pathway encompasses activation of protein conformational maintenance machineries, antioxidant enzymes, and metabolic changes simultaneously in the cytosol, mitochondria, and the ER. CORE induction extends replicative and chronological lifespan in budding yeast, highlighting its protective role against moderate proteotoxicity and its consequences such as the decline in respiration. Our findings accentuate that organelles do not function in isolation, but are integrated in a functional crosstalk, while also highlighting the importance of organelle communication in aging and age-related diseases.